Lignin, one of main components in the woody cell walls, is a complex heterogeneous biopolymer, which provides structural support and transportation of water in plants. It is highly recalcitrant to degradation (both chemically and environmentally) and protects cellulose from being degraded/hydrolyzed. Due to the structural complexity of native lignin, complete characterization and elucidation of lignin’s structure remains very challenging. The overarching goal of this work is to develop mass spectrometry based analytical methods to contribute to a better understanding of lignin structures. This dissertation will focus on the development and application of High-Resolution Accurate-Mass (HRAM) Mass Spectrometry (MS) as main analytical technique for studying lignin model compounds, including understanding the ionization behavior, studying corresponding fragmentation patterns and extracting structural information for structural elucidation eventually. Analytical methods were also developed to study the post-pretreatment products of the synthetic trimeric model compound using High-Performance Liquid Chromatography (HPLC) coupled with High-Resolution Accurate Mass (HRAM) Mass Spectrometry (MS). The first project of this dissertation focuses on mass spectral the characterization of lignin models from the in vitro oxidative coupling reactions. Three specific trimeric compounds were isolated and their ionization behaviors were investigated using HRAMMS via electrospray ionization (ESI). The reaction parameters of the in vitro oxidative coupling reaction were critical in alternating the linkage profiles of resulting dehydrogenation polymers (DHPs). Reaction parameters were tuned to obtain desired DHP linkages profile. Upon the isolation of three different trimeric compounds, a systematic comparison of ionization efficiency of three trimeric compounds was carried out using ESI-HRAM-MS under different ionization conditions. The second project was aimed to design a synthetic route for a lignin model compound that will be a good representation for native lignin during the pretreatment process. The model compound of interest has not been obtained previously through chemical synthesis. Due to the reactivity of cinnamyl alcohol, which contains the unsaturated side chain, this new synthesis strategy was developed based on the known aldol-type reaction route. A versatile synthesis procedure for preparation of β-O-4 oligomeric compounds was designed and implemented to include the most important functional groups (phenolic alcohol, aryl glycerol β-aryl ether bond and unsaturated side chain) in the resulting model compound. This new synthesis route also allowed incorporation of different monolignols. In the third project, Fenton chemistry was applied to a synthetic lignin model compound. Due to the non-specificity in the post pretreatment product profile, nontargeted analytical strategy was developed and applied to study the post-pretreatment products of the model compound using HPLC-HRMS. The results from this dissertation showed a significant difference in ionization behavior between three structurally different model compounds and indicated that primary structures of lignin compounds can largely affect corresponding electrospray ionization properties as well as fragmentation pattern. The work in this dissertation provides analytical techniques for non-targeted analysis of complex lignin samples and an insightful understanding of Fenton’s reaction pretreatment upon lignin model compound.

Mes travaux de thèse mettent en avant le développement de la stratégie métabonomique dans le cadre de deux thématiques d'actualité : le domaine du dopage sportif (Partie A) et celui de l'Exposome (Partie B). La première partie regroupe deux études et a pour objectif d'évaluer la contribution de la métabonomique au développement de nouveaux outils de criblage du dopage. Au cours de ces études, je me suis intéressée à l'analyse non-ciblée d'échantillons d'urine d'athlètes dopés et non- dopés, fournis par l'Agence Française de Lutte contre le Dopage et par des volontaires. L'originalité de cette démarche réside dans son caractère non-ciblé et plus particulièrement, dans sa capacité à mettre en évidence des perturbations au niveau métabolique grâce à (i) la spectrométrie de masse haute résolution (ToF) et très-haute résolution (FT-ICR) et (ii) l'analyse de données multivariées. Des potentiels biomarqueurs du dopage au tétrahydrocannabinol, salbutamol et budésonide ont ainsi pu être mis en évidence. La deuxième partie de ma thèse a pour objectif d'évaluer l'impact de la vinclozoline sur le système hormonal de rats et répond ainsi aux besoins de la nouvelle réglementation. Pour cette étude, je me suis donc intéressée aux échantillons de testicules de rats ayant subi un traitement à la vinclozoline. De par son caractère compréhensif, l'approche métabonomique m'a permis d'apporter des informations complémentaires aux études ciblées réalisées auparavant. Ces travaux me permettent alors de mettre en évidence les apports et les limites de la stratégie métabonomique par rapport : (1) au choix et à la préparation des échantillons d'origine biologique, (2) aux avantages et aux inconvénients des différentes techniques analytiques, (3) aux possibilités en termes de traitement des données, (4) aux exigences statistiques et (5) à la valeur biologique des résultats obtenus / My research work highlights the development of a metabonomic strategy through two topical issues: the doping in sport (Part A) and the Exposome (Part B). The first part includes two studies and aims to assess the contribution of metabonomics to the development of new screening tools. During these studies, I focused on the non-targeted analysis of clean and doped urine samples provided by the French Anti-Doping Agency and by volunteers. The originality of this approach lies in its non-targeted nature and, particularly, in its ability to highlight metabolic disruptions by (i) high-resolution (ToF) and very high-resolution (FT ICR) mass spectrometry and (ii) the analysis of multivariate data. The implemented strategy revealed several potential biomarkers for the use of tetrahydrocannabinol, budesonide and salbutamol. The second part of this thesis aims to evaluate the impact of vinclozolin on the hormonal system of rats and thus meets the requirements of the new regulations. For this study, I focused on testes extracts coming from rats treated with vinclozolin. Due to its comprehensive nature, the metabonomic study provided additional information to the previous targeted approach. All these results highlight the contributions and the limitations of metabonomics with regard to: (1) the choice and the preparation of biological samples, (2) the advantages and disadvantages of different analytical techniques, (3) the opportunities in terms of data processing, (4) the statistical requirements and (5) the biological value of the results

Due to increases in antibiotic resistance stemming from the overuse of commercial antibiotics, the need to discover novel antibacterial compounds is becoming more urgent. A species of Rhodococcus, MTM3W5.2, has been discovered and was found to produce a metabolite with inhibitory activity against closely related species. The aim of this study is to elucidate the structure of the inhibitory metabolite by isolating and purifying it; then characterizing it using spectroscopic techniques. The compound was isolated from MTM3W5.2 RM broth cultures using n-butanol extraction, which yielded an active crude extract. The crude extract was then subjected to fractionation using a Sephedex LH-20 column with a 100% methanol solvent. The inhibitory activity of the fractions was tested through disk diffusion assay using Rhodococcus erythropolis as an indicator. Further preparation was completed using preparative reverse-phase high-performance liquid chromatography. Advanced purification was conducted by multiple rounds of analytical reverse-phase HPLC. Throughout the study the HPLC fractions were characterized and stability was monitored using UV-Visible spectroscopy. Two pure samples at 58.xx and 72.xx minutes from HPLC collections were selected for further structural identification and are currently being studied using spectroscopic techniques, most notably 2D NMR

The advancements in the field of analytical chemistry, and especially mass spectrometry, have been redefining the field of contaminant detection. While more traditional analysis was sufficient to screen for a small number of well-known compounds, new techniques such as high-resolution mass spectrometry, have enabled a fairly comprehensive screening for previously unknown contaminants. This is enormously beneficial with respect to the analysis of water, air, or soil quality in a society that continuously introduces novel anthropogenic compounds into the environment. This dissertation, thus, focused on the analysis of the uncharacterized portion of compounds in two types of complex environmental matrices (i.e., crude oil and wastewater). First, targeted and non-targeted analyses were used in order to characterize a crude oil weathering series. Traditional techniques were used for the analysis of well-known oil components and led to the confirmation of biodegradation and photo-degradation trends. An ultrahigh-resolution mass spectrometric analysis was carried out in conjunction with several visualization plots in order to search for unknown compounds. While the study successfully detected a drastic increase in oxygenated components (likely ketones, quinones, and carboxylic acids), it also revealed severe limitations in the state of the art non-targeted crude oil analysis. Some of these limitations were explored in an in-depth atmospheric pressure ionization study of model petroleum compounds, and the dependence of ionization efficiency on numerous factors (e.g., size, heteroatom content, and methylation level) was shown. Since disregarding these intrinsic limitations leads to severely biased conclusions, these results provide crucial information for future crude oil characterization studies. Lastly, suspected and non-targeted analyses were used to evaluate contamination levels in wastewater-impacted interrelated water samples. A significant number of persistent compounds were tentatively identified, which represents an area of environmental concern that needs to be addressed further. Overall, this dissertation successfully applied non-targeted (in addition to targeted) analysis in order to screen for non-characterized compounds in crude oil and wastewater affected water samples. By doing so, the great potential of the growing field of non-targeted screening in order to expand the range of contaminants to include previously unknown and emerging compounds was highlighted.

Photodegradation is a major degradation mechanism for many environmental contaminants, both in indoor environments as well as in outdoor environments, and many of the contaminants are persistent and toxic. However, there is a lack of knowledge about the exact degradation process for many of them. It is important to not only monitor and study environmental contaminants that are commonly found. But it is also important to be able to identify their degradation products, however, due to lack of standards there is a difficulty to conduct identification. There is also the drawback with most spectral libraries used for identification being recorded at low resolution, when new instruments are being developed for using high resolution. This makes it challenging to identify compounds with spectral peaks that differ only slightly in their mass-to-charge ratio (m/z). Thus, the aim of this study was to determine potential degradation products from different compounds commonly found in indoor environments and then add these findings to a spectral library for high resolution mass spectrometry. This was conducted by first exposing the standards to ultraviolet (UV) light, and then the chemical analysis was performed using a gas chromatography coupled with a high resolution mass spectrometer. All data was then processed in different software in order to determine potential products. Many of the compounds did not indicate any degradation using the experimental settings, and for some not even the parent compound could be identified. In total, four potential degradation products were found in this study; degradation products were identified for hexabromobenzene (HBB) with penta-, tetra-, and tribromobenzene, as well as one potential product for 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EHTBB).

<p>Human activities represent a significant burden on the global water cycle, with large and increasing demands placed on limited water resources by manufacturing, energy production and domestic water use. In addition to changing the quantity of available water resources, human activities lead to changes in water quality by introducing a large and often poorly-characterized array of chemical pollutants, which may negatively impact biodiversity in aquatic ecosystems, leading to impairment of valuable ecosystem functions and services. Domestic and industrial wastewaters represent a significant source of pollution to the aquatic environment due to inadequate or incomplete removal of chemicals introduced into waters by human activities. Currently, incomplete chemical characterization of treated wastewaters limits comprehensive risk assessment of this ubiquitous impact to water. In particular, a significant fraction of the organic chemical composition of treated industrial and domestic wastewaters remains uncharacterized at the molecular level. Efforts aimed at reducing the impacts of water pollution on aquatic ecosystems critically require knowledge of the composition of wastewaters to develop interventions capable of protecting our precious natural water resources.</p><p>The goal of this dissertation was to develop a robust, extensible and high-throughput framework for the comprehensive characterization of organic micropollutants in wastewaters by high-resolution accurate-mass mass spectrometry. High-resolution mass spectrometry provides the most powerful analytical technique available for assessing the occurrence and fate of organic pollutants in the water cycle. However, significant limitations in data processing, analysis and interpretation have limited this technique in achieving comprehensive characterization of organic pollutants occurring in natural and built environments. My work aimed to address these challenges by development of automated workflows for the structural characterization of organic pollutants in wastewater and wastewater impacted environments by high-resolution mass spectrometry, and to apply these methods in combination with novel data handling routines to conduct detailed fate studies of wastewater-derived organic micropollutants in the aquatic environment. </p><p>In Chapter 2, chemoinformatic tools were implemented along with novel non-targeted mass spectrometric analytical methods to characterize, map, and explore an environmentally-relevant “chemical space” in municipal wastewater. This was accomplished by characterizing the molecular composition of known wastewater-derived organic pollutants and substances that are prioritized as potential wastewater contaminants, using these databases to evaluate the pollutant-likeness of structures postulated for unknown organic compounds that I detected in wastewater extracts using high-resolution mass spectrometry approaches. Results showed that application of multiple computational mass spectrometric tools to structural elucidation of unknown organic pollutants arising in wastewaters improved the efficiency and veracity of screening approaches based on high-resolution mass spectrometry. Furthermore, structural similarity searching was essential for prioritizing substances sharing structural features with known organic pollutants or industrial and consumer chemicals that could enter the environment through use or disposal.</p><p>I then applied this comprehensive methodological and computational non-targeted analysis workflow to micropollutant fate analysis in domestic wastewaters (Chapter 3), surface waters impacted by water reuse activities (Chapter 4) and effluents of wastewater treatment facilities receiving wastewater from oil and gas extraction activities (Chapter 5). In Chapter 3, I showed that application of chemometric tools aided in the prioritization of non-targeted compounds arising at various stages of conventional wastewater treatment by partitioning high dimensional data into rational chemical categories based on knowledge of organic chemical fate processes, resulting in the classification of organic micropollutants based on their occurrence and/or removal during treatment. Similarly, in Chapter 4, high-resolution sampling and broad-spectrum targeted and non-targeted chemical analysis were applied to assess the occurrence and fate of organic micropollutants in a water reuse application, wherein reclaimed wastewater was applied for irrigation of turf grass. Results showed that organic micropollutant composition of surface waters receiving runoff from wastewater irrigated areas appeared to be minimally impacted by wastewater-derived organic micropollutants. Finally, Chapter 5 presents results of the comprehensive organic chemical composition of oil and gas wastewaters treated for surface water discharge. Concurrent analysis of effluent samples by complementary, broad-spectrum analytical techniques, revealed that low-levels of hydrophobic organic contaminants, but elevated concentrations of polymeric surfactants, which may effect the fate and analysis of contaminants of concern in oil and gas wastewaters. </p><p>Taken together, my work represents significant progress in the characterization of polar organic chemical pollutants associated with wastewater-impacted environments by high-resolution mass spectrometry. Application of these comprehensive methods to examine micropollutant fate processes in wastewater treatment systems, water reuse environments, and water applications in oil/gas exploration yielded new insights into the factors that influence transport, transformation, and persistence of organic micropollutants in these systems across an unprecedented breadth of chemical space.</p> / Dissertation

The characterization of lipids is performed by mass spectrometry based on structure specific fragments or by accurate mass measurements of intact precursor ions. The latter method, termed ’top-down lipidomics’, is due to its robustness, simplicity and speed a valuable tool for medical research to elucidate the molecular background of lipid metabolic disorders. The current thesis aims to improve the established lipidomics methods. Therefore, a new top-down lipidomics method was introduced that increased the analysis throughput, lipidome coverage and accuracy of quantification, compared to previous approaches, by rapid successive acquisition of high resolution Fourier transform mass spectra in positive and negative ion modes. Furthermore, the characterization of molecular lipid species by utilizing high energy collisional dissociation was achieved on Orbitrap instruments. The mass accuracy of acquired MS/MS spectra increased the confidence in identification for unusual very-long chain polyunsaturated phosphatidylcholine species and a new lipid class, the maradolipids. Beyond that, effort was made to enhance the accuracy and comparability of MS/MS based bottom-up lipidomics data. In this respect, lipids with varying degree of unsaturation were analyzed and revealed discrete fragmentation properties. The technical refined lipidomics methods allowed insight into the lipid composition of lipoproteins and changes of the blood plasma induced by apheresis. Lipidomics screening of blood plasma uncovered an altered lipid pattern in consequence of impaired glucose metabolism and type 2 diabetes. The lipidomics characterization of islet allowed their quality assessment.

Orientador: Marcos Nogueira Eberlin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T13:56:59Z (GMT). No. of bitstreams: 1 Klitzke_ClecioFernando_D.pdf: 5751557 bytes, checksum: 0cd80e6f5c01aa04c7fdd89f600b374c (MD5) Previous issue date: 2012 / Resumo: O petróleo é uma mistura complexa, constituída de milhares de compostos e sua caracterização é fundamental em diversos processos da indústria petrolífera. A abordagem moderna para a caracterização total dos compostos polares (N, O, S) do petróleo é através da petroleômica, utilizando a espectrometria de massas de altíssima resolução e exatidão obtida em espectrômetros de ressonância ciclotrônica de íons com análise por transformada de Fourier (FT-ICR MS). FT-ICR MS é o equipamento ideal para estas análises, no entanto a literatura carece de informações quanto a otimização do número de scans e do efeito da resolução na análise petroleômica. Determinamos como condições ideais para a análise destas amostras de altíssima complexidade a obtenção de espectros de MS com poder de resolução de 400.000 em m/z 400 e acúmulo de 100 mscans. Foram analisados também os compostos polares presentes em outras fontes de combustíveis fósseis, como carvão mineral e folhelho. Os resultados mostraram padrões distintos em relação à origem do material e possivelmente ao grau de evolução térmica. Comparamos a análise por FT-ICR MS dos ácidos carboxílicos de amostras de petróleo com os resultados das análise de frações ácidas por FAB MS obtidas no CENPES . PETROBRAS, mostrando que as análises por FT-ICR MS apresentam resultados superiores na identificação da distribuição de DBE sendo que por FAB MS não temos informações relativas a DBE superior a 7. A análise abrangente da composição de ácidos do petróleo é de extrema importância com conta da corrosão e formação de depósitos e emulsões, dependentes da composição destes ácidos. Comparamos alguns resultados da análise por FT-ICR MS com TOF MS (UHRT) de altíssima resolução, 100.000. Os resultados obtidos com UHRT MS estão próximos as análises por FT-ICR MS com 200.000 de resolução em m/z 400. Obtivemos a identificação de boa parte das classes de heteroátomos úteis nas análises petroleômicas. Analisamos por Travelling Wave Ion Mobility Mass Spectrometry (TWIM MS) padrões de ácidos, destilados e amostras de petróleo. Os resultados obtidos mostraram uma tendência da redução do drift time com o aumento da DBE dos analitos. As amostras de petróleo também revelaram perfis distintos para diferentes faixas de drift time, sendo possível diferenciar classes de compostos polares (O2, N e NO). Os resultados obtidos mostraram o grande potencial da análise por FT-ICR MS e UHRT MS, complementados pela análise estrutural fornecida pela mobilidade iônica com o uso da TWIM MS. / Abstract: Crude oil is a complex mixture consisting of thousands of compounds and their characterization is essential in various processes of the petroleum industry. The modern approach to the characterization of total polar compounds (N, O, S) of oil is through petroleomics MS, using the ultra-high resolution mass spectrometry and accuracy obtained in ion cyclotron resonance spectrometers with analysis by Fourier transform (FT-ICR MS). FT-ICR MS is the ideal equipment for these analyses, however the literature lacks information about the optimization of scan number and the effect of resolving power in the petroleomic analysis. We determine the ideal conditions for the analysis of these samples of high complexity getting MS spectra with resolving power of 400,000 in m/z 400 and accumulation of 100 mscans. Polar compounds in crude oil, and other sources of fossil fuels such coal and shale were analyzed. The results showed distinct patterns in relation to the origin of the material and possibly the degree of thermal evolution. We compare the performance of FT-ICR MS with FAB MS in the analysis of carboxylic acids composition of crude oil samples. Analyses by FT-ICR MS feature superior results in identifying the distribution of DBE. FAB MS does not have information on DBE exceeding 7. Comprehensive analysis of the composition of crude oil acids is of extreme importance with account of corrosion and formation of deposits and emulsions, dependent on the composition of these acids. We compare some results of the analysis by FT-ICR MS with ultra-high resolution TOF MS (UHRT) with 100,000 of resolving power. The results obtained with UHRT MS are equivalent to the FT-ICR MS with 200,000 resolving power at m/z 400. We obtained the identification of most of the classes of heteroatoms useful in petroleomic analysis and the results show that UHRT performance is near to FTICR. Traveling Wave Ion Mobility Mass Spectrometry (TWIM MS) show good results to study the patterns of ion mobility of acid samples, distillates cuts and crude oil samples. The results showed a trend of reducing the drift time with increasing DBE of the analytes. The crude oil samples also revealed different drift time profiles for different classes of polar compounds (O2, N, and NO). The results obtained show the great potential of FT-ICR MS and UHRT MS analysis, complemented by structural analysis provided by ion mobility with the use of TWIM MS, of distillate cuts and crude oil samples. / Doutorado / Quimica Organica / Doutor em Ciências

There are a great variety of environmentals contaminants, a lot of which are understudied asit is today. The aim of this study was to develop an in-house high-resolution spectral librarycontaining environmental contaminants, with focus on plastic additives. The spectral librarywould then be used to perform suspect screening on real sample extracts. A list of standardswas acquired, prepared and then analyzed with GC-EI-Orbitrap. The files from the analysiswere converted and then processed in a software called MS-DIAL. Once the analyte wasidentified, the data was exported to MS-FINDER. MS-FINDER performs fragmentannotation and generates in silico spectra, and the spectral data is then saved as an MSP file.An MSP file is a file based on text containing mass spectrometric data, and is the mostcommonly supported file for spectral libraries. The individual MSP files for the standards arethen collected into a common MSP file, which can be opened in MS-DIAL to performsuspect screening. The suspect screening in this study was performed as a retrospectiveanalysis of indoor dust samples from a study in 2021.From the beginning, 60 standards out of 80 available standards were included. The principalreason for a standard not being included was that the compound would most certainly notelute using GC with the set analytical parameters. Out of the 60 analyzed standards, 32compounds could be added to the spectral library. No distinct peak within thechromatographic run time was the main reason for not being able to add a standard. Despiteadding 32 different compounds into the spectral library, a total of 51 individual MSP-fileswere generated. Some of the technical mixtures generated a chromatogram with severaldifferent peaks, and MSP files were generated for each of the most prominent peaks. Thiswas done since it could still provide useful information in an identification process.The suspect screening in this study was performed as a retrospective analysis of indoor dustsamples from 2019. Into the spectral library, 32 different compounds were included, and 10of these were found in the different indoor dust samples when performing the suspectscreening.Building a spectral library in this way is a time consuming process with a lot of manual work,but spectral libraries are nonetheless necessary in the process of effectively monitoringenvironmental contaminants, to assist in risk assessment and decision making for regulatorson identified contaminants.

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