| Ambient ionization techniques allow complex samples to be analyzed in their native state with great advantages including minimal sample preparation, simplicity, high speed, and versatility to mass spectrometry. Clear visualization of molecules inside bulk samples with high chemical complexity has challenged current ambient ionization techniques and is of increasing importance in multiple disciplines such as clinical diagnosis, plant science, catalyst studies, materials science, etc. In routine analytical techniques, multi-step sample pre-treatment is inevitable prior to profiling of endogenous chemicals inside a bulk tissue.Recently, the young family of ambient pressure ionization mass spectrometry allows for direct analysis of complex samples with no pre-treatment requirement. However, current ambient ionization methods mostly focused on surface analysis of intact bulk sample. Thus, rendering a method capable of characterizing inherent chemicals in the bulk volume is preferably demanded. Herein we report a novel strategy termed as internal extractive electrospray ionization(i EESI) for the straight-forward MS analysis of whole-volume samples without either mashing/grinding the sample or matrix clean-up. The method allows both qualitative and quantitative analysis of analytes distributed in a 3-dimensional volume of various synthetic and biological matrices. The presented method is featured by the high speed of analysis, high sensitivity, low sample consumption and minimal sample preparation and/or degradation, offering unique possibilities for the applications in plant science, clinical diagnosis, catalyst studies, and materials science.Based on the brief overview of the principle and applications of ambient ionization techniques, the main contents of this thesis are as follows:1. The operation, parameter setting, working principle, and technical characteristics of internal extractive electrospray ionization mass spectrometry(i EESI-MS) were studied.2. i EESI-MS was applied for the study of plant metabolomics for direct in vivo profilingof phytochemicals in various native plant tissues including leaves, roots and fruits. By passing electrospray solvent through a plant tissue sample, various metabolites including amino acids,sugars, antioxidants and alkaloids were continuously extracted from interior plant tissue and directly ionized for mass spectrometric analysis. Phytochemical patterns obtained by i EESI-MS allowed rapid differentiation of various species of a plant(e.g., ginkgo), as well as characterization of physiological/pathologic conditions(e.g., chlorophytum comosum) and maturity stage(e.g., strawberries). Our results demonstrate that i EESI-MS approach is potentially useful for in vivo phytochemomics studies with minimal biodegradation, allowing characterization of plant metabolism with molecular sensitivity, high speed and simplicity of analysis.3. The applications of i EESI-MS in food quality and food safity analysis were preformed.1) i EESI-MS has been extended to direct molecular analysis of garlic. By obviating time-consuming sample preparations, chemicals including organosulfur compounds, amino acids and saccharide were successfully detected and identified via collision-induced dissociation(CID). Mass spectral fingerprints of different kinds of garlic cloves, as well as various post-treatment ones, were further processed for principal component analysis(PCA)to better visualize the differences. Our experimental results indicate that i EESI-MS allows rapid quality recognition of garlic bulbs. 2) i EESI-MS was employed to directly analyse the choline, fructose and sucrose in four kinds of red peppers under positive ion detection mode in the mass range of m/z 50~1000. Changes of the chemical constituents in the red pepper during long time storage(12 days) were directly followed using i EESI-MS, and PCA was used to visualize the difference of red peppers treated with different storage period. The result indicates that the relative abundance ratio of choline to fructose and the relative abundance ratio of sucrose to fructose in red peppers was linearly decrease during the storage, and the corresponding decline curves which associate with the metabolism of the red pepper were plotted. Based on the ratio of choline/fructose or sucrose/fructose, the freshness and quality of the red pepper could be identified by i EESI-MS at the molecular level. 3) Representive specimen of meat samples including pig lung, pig lean meat and fat meat were analyzed using i EESI-MS. Suspected signals of illegial additive of targeted “β-agonists” components in mass spectrometric window were isolated for CID experiments to avoid false positives results.β-agonists including clenbuterol and procaterol in pork samples were rapidly detected witnin20 s, and the samples consumption of IEESI-MS is also less than 100 mg. Overall, i EESI-MS shows the merit of such as minimum sample pretreatment, low sample consumption, and rapid analysis, showing promising applications in food quality control analysis and food safety analysis.4. Chemical characterization of ongoing enzymatic reactions occurring inside living garlic samples has been demonstrated using internal extractive electrospray ionization massspectrometry(i EESI-MS). Both large molecules(e.g., polysaccharides) and small molecules(e.g., amino acids, alkaloids and organosulfur compounds, etc.) were successfully detected with high abundances from the interior of raw garlic cloves without sample pretreatment. The essential components(e.g., alliin, and allicin) involved in the enzymatic conversion of alliin were monitored in real time, showing that i EESI-MS is capable to characterize the dynamic metabolic response to external stimuli including high and low temperature, microwave and ultrasound irradiation, and exposure to extreme p H values. The method established here has been further applied to molecular profiling of the germination process in garlic cloves,providing the metabolic profiles at different sprouting stages. The experimental results demonstrate that i EESI-MS can be a powerful method to probe the ongoing enzymatic reactions occurring in untreated biological samples and to allow deeper mechanistic understanding of enzyme properties in native biological environment.5. A facile method for high throughput quantification of 1-hydroxypyrene(1-OHP, the most widely used biomarker of internal dose of PAHs exposure) in undiluted human urine samples was established by using magnetic solid-phase extraction coupled with internal extractive electrospray ionization tandem mass spectrometry. Based on previous research,MSPE absorbents of polypyrrole-coated Fe3O4 magnetite nanocomposites was selected to selectively enrich trace 1-OHP in undiluted human urine samples, and then directly eluted by the electrospraying solvent biased with high voltage to produce the deprotonated 1-OHP molecules for mass spectrometric analysis. The method established here significantly improved the performance for detection of urinary 1-OHP. Based on this proposed method,practical urine samples from people with various PAHs exposure including lung cancer patients and other high-risk occupation groups were studied using MSPE-i EESI-MS. The results demonstrated that MSPE-i EESI-MS is a useful tool for rapid quantification of urinary1-OHP for health risk assessment of PAHs exposure. |
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