La composition chimique de la résine sandaraque et de son composant principal l’acide communique a été étudiée par chromatographie en phase gazeuse – spectrométrie de masse (GC-MS), MALDI-TOF (désorption-ionisation laser assistée par matrice - temps de vol), ESI (ionisation par électronébuliseur) - Orbitrap, FTIR/ATR (spectroscopie infrarouge à transformée de Fourier/réflectance totale atténuée), spectroscopie de RMN (résonance magnétique nucléaire) à l'état solide et liquide. Six composés avec des squelettes labdane et pimarane ont été identifiés dans la résine commerciale. Les spectres de masse obtenus ont été interprétés et le comportement en spectrométrie de masse de ces diterpénoïdes dans les conditions de l’impact électronique a été décrit. L'analyse quantitative par la méthode de l'étalon interne a révélé que les diterpénoïdes identifiés ne représentaient que 10 à 30% de l'échantillon analysé. La complexité de la fraction réticulée de la résine commerciale sandaraque est bien reflétée par les spectres de masse MALDI-TOF et ESI-Orbitrap. En conséquence, les spectres de masse de MALDI-TOF comprenaient trois clusters de pics dans la gamme m/z de 300-900, et ceux d’ESI-Orbitrap contenaient cinq clusters de pics dans la gamme m/z de 300-1100. Les pics dans les clusters correspondent aux dérivés oxygénés des diterpénoïdes. Les résultats obtenus à partir des expériences RMN par IRCP (Inversion Recovery Cross-Polarization) ont révélé le caractère rigide des échantillons de la résine sandaraque analysés et justifiaient l'hypothèse que le reste de l'échantillon, qui ne pouvait être quantifié par la méthode de l'étalon interne, aurait un caractère polymère. / The chemical composition of sandarac resin and its principal component communic acid was investigated by gas chromatography-mass spectrometry (GC-MS), MALDI-TOF (Matrix Assisted Laser Desorption Ionization - Time of Flight), ESI (Electrospray ionization)-Orbitrap, FTIR/ATR (Fourier transform infrared spectroscopy/Attenuated total reflectance), liquid- and solid state NMR (Nuclear magnetic resonance) spectroscopy. Six compounds with labdane and pimarane skeletons were identified in the commercial resin. The obtained mass spectra were interpreted and the mass spectrometric behaviour of these diterpenoids under EI conditions was described. Quantitative analysis by the method of internal standard revealed that identified diterpenoids represent only 10–30% of the analysed sample. The complexity of the reticulated fraction of the commercial sandarac resin was well reflected by the MALDI-TOF and ESI-Orbitrap mass spectra. As a result, MALDI-TOF mass spectra comprised three clusters of peaks in the m/z range of 300–900, and for the ESI-Orbitrap mass spectra contained five clusters of peaks in the m/z range of 300–1100. The peaks in the clusters corresponded to the oxygenated derivatives of the diterpenoids. The results obtained from the IRCP (Inversion Recovery Cross-Polarization) experiments revealed the rigid character of the sandarac resin samples analyzed and justified the hypothesis that the rest of the sample, which could not be quantified by the method of internal standard, would have a polymeric nature.

Ring closing metathesis (RCM) is a chemical transformation that converts a bisalkene compound into a cycloalkene. It is catalyzed by transition metal complexes containing carbene ligands (that feature metal-carbon double bonds). The mechanism is well-understood, however, there are numerous details of the reaction that are less well understood, especially concerning catalyst activation and decomposition and formation of byproducts. This thesis takes a new approach to the study of RCM: analysis of the reaction using real-time mass spectrometric techniques. Electrospray ionization (ESI) mass spectrometry was employed in this study, and the real-time aspect was enabled by using pressurized sample infusion (PSI). Observation of the reactants and products was enabled using charge-tagged bis-alkenes of the general formula [Bu2N{(CH2)nCH=CH2}2]+ [PF6]–. These were synthesized in two steps using a generally applicable methodology to generate a wide range of ring sizes of the product, from 5- to 15-membered rings. Examination of their behavior under carefully optimized RCM conditions using Grubbs’ second-generation catalyst showed a wide variation in reaction rates and amount of byproducts, largely due to ring-strain effects (especially high for 5- and 9-membered rings). Byproducts always exhibited a 14 Da mass unit difference from starting materials or products, and Orbitrap MS analysis confirmed it was CH2. Isomerization was suspected to lead to byproducts. A pathway for byproducts via isomerization and cross metathesis was proposed. The source of actual isomerization catalyst was believed to be from the precatalyst itself as the evidence of precatalyst decomposition was observed. Finally, to prove our isomerization hypothesis, an authentic isomerization catalyst was deliberately added into a fast and clean reaction along with Grubbs’ second-generation catalyst, and it produced the expected byproducts. Only small amounts of oligomeric intermediates were observed, probably because of the low concentrations used. [ClPCy3]+ was a new short-lived decomposition product stemming from catalyst breakdown, along with already-known imidazolium and protonated phosphine decomposition products. Overall, the thesis provides deep new insights into the nature of RCM reactions, in particular revealing the importance of isomerization in RCM reactions that are slow due to ring strain effects and in uncovering a new decomposition pathway for important RCM catalysts. / Graduate

Ces travaux de thèse concernent les développements d'une méthode de séparation chromatographique couplée simultanément à l'ESI-MS et l'ICP-MS afin de réaliser l'analyse de spéciation exhaustive des lanthanides en phase aqueuse représentative des phases de désextraction des procédés de traitement du combustible usé. Cette méthode analytique permet de séparer, caractériser et quantifier des complexes de lanthanides à ligands polyaminocarboxyliques comme le DTPA et l'EDTA, utilisés comme agents complexants dans ces procédés. La méthode de séparation par chromatographie HILIC des complexes de lanthanides a été mise au point avec la phase stationnaire à fonctions amide. Un criblage d'une large gamme de compositions de phase mobile a permis de déterminer que le mécanisme d'adsorption est prédominant lors l'élution des complexes de lanthanides et d'obtenir des conditions de séparation optimisées. Des conditions d'analyse plus rapides obtenues avec une colonne à fonctions amide de granulométrie sub-2 µm et de longueur plus faible ont permis de réduire le temps d'analyse d'un facteur 2,5 et la consommation de solvant de 25 %. La caractérisation structurale et isotopique par HILIC ESI-MS a été réalisée ainsi que la mise au point d'une méthode d'étalonnage externe. Les performances analytiques de la méthode de quantification ont été déterminées. Enfin, le développement d'un système de couplage de l'HILIC à l'ESI-MS et l'ICP-MS a été réalisé. Une méthode de quantification simultanée par ESI-MS et par ICP-MS a permis de déterminer la distribution quantitative des espèces en solution ainsi que les performances analytiques associées. / This work focuses on the development of a chromatographic separation method coupled to both ESI-MS and ICP-MS in order to achieve the comprehensive speciation analysis of lanthanides in aqueous phase representative of back-extraction phases of advanced spent nuclear fuel treatment processes. This analytical method allowed the separation, the characterization and the quantitation of lanthanides complexes holding polyaminocarboxylic ligands, such as DTPA and ETDA, used as complexing agents in these processes. A HILIC separation method of lanthanides complexes has been developed with an amide bonded stationary phase. A screening of a wide range of mobile phase compositions demonstrated that the adsorption mechanism was predominant. This screening allowed also obtaining optimized separation conditions. Faster analysis conditions with shorter amide column packed with sub 2 µm particles reduced analysis time by 2.5 and 25% solvent consumption. Isotopic and structural characterization by HILIC ESI-MS was performed as well as the development of external calibration quantitation method. Analytical performances of quantitation method were determined. Finally, the development of the HILIC coupling to ESI-MS and ICP-MS was achieved. A simultaneous quantitation method by ESI-MS and ICP-MS was performed to determine the species quantitative distribution in solution. Analytical performances of quantitation method were also determined.

Plant estrogens have long been associated with health benefits. The potential of tissue culture techniques for the production of several secondary metabolites has been known for many years. Tissue cultures stimulate the production or induce the biosynthesis of novel compounds not found in the mature plant. Tissue culture of Morus alba, family Moraceae, is known to contain phytoestrogens, was established on plant-hormone supplemented Murashige and Skoog (MS) medium. Petiole and the stem tissue from mature trees were the best explants for initiation and proliferation of calli. The best callus proliferation was obtained on MS medium containing 1-napthalene acetic acid (1mg/ml) and benzylaminopurine (0.5mg/ml) for M. alba. Comparison of phytoestrogens of Moraceae species from in vivo and in vitro tissue isolation were carried out. The estrogenic activities of callus extracts were assayed in an estrogen-responsive yeast system expressing the human estrogen receptor alpha. Male callus extracts had higher estrogenic activity than male and female extracts from in vivo and in vitro tissues. Isolation and characterization of phytoestrogens from above tissues were carried out using solid phase extraction, high perfomance liquid chromatography and mass spectrometry techniques. Biochanin A, an isoflavonoid, was isolated as one of the compounds in male callus extracts. Biochanin A has been known to have an antiestrogenic acitivity in mammals. Isoflavonoid compounds have been characterized as strong protein tyrosine kinase inhibitors in variety of animal cells. Isoflavones are structurally similar to estradiol, and display agonistic and antagonistic interactions with the estrogen receptor. Isoflavones possess therapeutic and preventive properties such as being used for postmenopausal osteoporosis, breast cancer, and inhibition of tumors.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-08-04T13:34:48Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE - Waleska de Araújo Siqueira Bento - Versão final.pdf: 3392903 bytes, checksum: 633a749c72e4eb3f1d0e4f6b0f0c0b83 (MD5) / Made available in DSpace on 2017-08-04T13:34:48Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE - Waleska de Araújo Siqueira Bento - Versão final.pdf: 3392903 bytes, checksum: 633a749c72e4eb3f1d0e4f6b0f0c0b83 (MD5) Previous issue date: 2016-03-11 / O desenvolvimento de métodos de preparo de amostras é de suma importância para a análise química. Ao longo dos anos, cada vez mais os cientistas buscam técnicas para aprimorar metodologias e ferramentas matemáticas para a validação dos métodos desenvolvidos. No presente trabalho foram desenvolvidos dois diferentes métodos de preparo de amostras: um para a análise de corantes artificiais em iogurtes e posterior análise por HPLC-PAD e outro método utilizando a técnica de CE-UV/Vis para separar e fracionar amostras padrões de misturas de proteínas, seguido de um método para digestão destas e análise por LC-ESI-MS para posterior mapeamento dos peptídeos. O primeiro trabalho foi realizado para determinar a concentração de corantes artificiais em iogurtes e bebidas lácteas que são alimentos produzidos a partir da fermentação do leite sendo um alimento importante e indispensável a dieta de adultos e crianças. Para tanto foi desenvolvido um método de extração e determinação de corantes artificiais em iogurtes e bebidas lácteas por HPLC-PAD. O método foi devidamente validado e as amostras comerciais analisadas estavam de acordo com a legislação. Já o segundo trabalho visa o estudo da proteômica através do mapeamento de peptídeos. A eletroforese capilar (CE) por ser uma técnica de separação muito eficiente para a análise de proteínas e peptídeos foi utilizada na etapa inicial de preparo da amostra. A separação inicial foi seguida do fracionamento da amostra que é uma etapa muito importante e que a CE pode proporcionar, minimizando a complexidade da amostra. Posteriormente foi desenvolvido um método para a digestão das proteínas contidas em cada fração e o mapeamento dos peptídeos foi realizado com auxílio do LC-ESI-MS. Os resultados obtidos são animadores, visto que foi possível digerir as proteínas com quimotripsina em até duas horas. O que é um tempo curto se comparado a trabalhos da literatura que necessitaram de 12 a 24 horas para digestão de proteínas quando utilizadas enzimas livres em solução. / The development of sample preparation methods is of paramount importance for analytical chemistry. Over the years, more and more scientists are seeking techniques to improve methodologies and mathematical tools for the validation of the methods they have developed. In this study two different methods of sample preparation were developed: one for analysis of artificial colorants in yogurts and subsequent analysis by HPLC-PAD; and another method using the CE-UV/Vis technique to separate and fractionate standard samples of protein mixtures, followed by a digestion method for these and analysis by LCESI-MS for subsequent peptides mapping. The first study was conducted to determine the concentration of artificial colorant in yoghurts and milk drinks, foods produced from the fermentation of milk that are an important and indispensable part of the food diet for adults and children. To this end, a method was developed for the extraction and determination of artificial colorant in HPLC-PAD yoghurts and milk drinks. The method was validated and commercial samples were analyzed according to the legislation. The second work is aimed at the study of proteomics by peptide mapping. Capillary electrophoresis (CE) as an effective separation technique was used in the initial stage of sample preparation for the anaysis of proteins and peptides. The initial separation was followed by fractionation of the sample which is a very important step enabled by CE, thus minimizing sample complexity. Then, a method was developed to digest the proteins contained in each fraction and the mapping of the peptides was carried out with the aid of LC-ESI-MS. The results obtained are encouraging, since the study showed that it was possible to digest proteins with free chymotrypsin within two hours. This is a short time compared to that found in the published papers requiring 12 to 24 hours for protein digestion when free enzymes in solution are used.

No description available.

Over the past decades, electrospray ionization for mass spectrometry (ESI-MS) has become one of the most commonly employed techniques in analytical chemistry, mainly due to its broad applicability to polar and semipolar compounds and the superior selectivity which is achieved in combination with high resolution separation techniques. However, responsiveness of an analytical method also determines its suitability for the quantitation of chemical compounds; and in electrospray ionization for mass spectrometry, it can vary significantly among different analytes with identical solution concentrations. Therefore, we investigated the ESI-response behavior of 56 nitrogen-containing compounds including aromatic amines and pyridines, two compound classes of high importance to both, synthetic organic chemistry as well as to pharmaceutical sciences. These compounds are increasingly analyzed employing ESI mass spectrometry detection due to their polar, basic character. Signal intensities of the peaks from the protonated molecular ion (MH+) were acquired under different conditions and related to compound properties such as basicity, polarity, volatility and molecular size exploring their quantitative impact on ionization efficiency. As a result, we found that though solution basicity of a compound is the main factor initially determining the ESI response of the protonated molecular ion, other factors such as polarity and vaporability become more important under acidic solvent conditions and may nearly outweigh the importance of basicity under these conditions. Moreover, we show that different molecular descriptors may become important when using different types of instruments for such investigations, a fact not detailed so far in the available literature.

Wax esters are substantial constituents of natural waxes, which can be found in many living organisms. Properties of lipids, including wax esters, may be significantly influenced by the position of double bond. In this diploma thesis the location of double bonds was determined by dimethyl disulfide (DMDS) derivatization followed by detection using tandem mass spectrometry with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). We managed to measure the APCI and ESI MS/MS spectra of 8 different wax esters with different position of double bond. Diagnostic ions determining double bond position were identified. This method could be used in HPLC/MS analysis of wax esters, which cannot be analyzed by GC/MS. It was shown that the DMDS derivatization reaction and mass detection with APCI ionization is also suitable for locating double bonds in alkenes.

No description available.

No description available.