<p> Quantitation by nuclear magnetic resonance (NMR) has been frequently done with integrals. The use of peak heights has been thought to be unreliable. The aim of this thesis is to examine the reliability of peak height method in achieving quantitative NMR measurements for small molecules such as water and sucrose.</p> <p> Isotope measurements have been traditionally done by isotope-ratio mass spectrometry. This is no doubt a highly sensitive technique for analyzing pure samples but the analysis of mixtures is not as straight-forward as it would be for the former. Ontario Hydro has encountered problems in measuring deuterium in DMSO/water mixtures. To solve the problem with NMR, an analytical method has been established to measure the deuterium content in waters and in DMSO/water mixtures. This involved testing a linear model for analyzing waters which were enriched or depleted with deuterium as well as applying the model to quantify DMSO/water mixtures. Both 1H and 2H NMR were employed. Satisfactory accuracy and precision of the results were obtained.</p> <p> For quantitative 13C work, the peak height method is often not recommended due to the variations in signal width, which is a result of varied T2 values and nuclear Overhauser enhancement (NOE). Sucrose molecules in cane sugar and beet sugar have different 13C isotopic ratios because they are synthesized by different photosynthetic pathways. To see the usefulness and limitation of the peak height method, 13C spectra of sucrose were acquired and the carbon peaks were quantified. Good precision was achieved but no predictable trend in the isotope difference could be found.</p> / Thesis / Master of Science (MSc)

Solution nuclear magnetic resonance (NMR) spectroscopy is a valuable analytical technique that is nondestructive, highly reproducible, and relatively quick to identify and quantify many chemical compounds. Quantitative NMR is a technique commonly used in many medical applications such as drug analysis, metabolomics, and protein-nanoparticle (P-NP) interactions. The most common technique used is the proton (1H) NMR experiment. The 1H NMR analysis provides a quick snapshot of the interested compounds in solution. However, as the compounds become more complex the spectrum becomes overpopulated. This dissertation focuses on various quantitative NMR techniques applied to metabolic and protein competition studies. Specifically, we investigated the effect of biochar on Escherichia coli (E. coli) growth to provide insight on how the metabolic pathways were influenced with the addition of biochar in the RPMI media. A 1H NMR spectrum was recorded at various time points to monitor the metabolic changes over time as E. coli grew in the presence and absence of biochar. The spectra were compared to an in-house metabolite library to identify and quantify the metabolic changes in E. coli. To enhance our metabolic library analysis, we utilized a pure shift analysis attached to the TOCSY pulse program to deconvolute spin systems by using a second dimension for analysis. DIPSI-PSYCHE TOCSY was applied to investigate a metabolite mixture sample and Streptococcus pneumoniae (S. pneumoniae) extracellular metabolites to better resolve the spin systems that significantly overlap each other in the 1H NMR spectra. Our novel approach suggests that adding a pure shift to the TOCSY pulse program is extremely beneficial to investigate various metabolic profiles. Finally, we investigated the protein competition to the AuNP surfaces using a 2D 1H-15N HSQC pulse program. Specifically, we used 1H-15N HSQC technique to quantify the binding capacity for each protein to the AuNP surface before we investigated the competition of two proteins, GB3-Ubq (model protein mixture) or AM-R2ab (biofilm forming protein mixture) to the surface. We also employed a model to study the kinetics of the protein competition to the surface. Our model suggests that GB3-Ubq does not specifically behave kinetically but AM-R2ab is strictly kinetically controlled.

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Die Globalisierung von Handel und Wirtschaft macht es nötig, nationale Analysenergebnisse international gegenseitig anzuerkennen. Dabei kann die Richtigkeit der Analysenwerte durch Ruckführung auf Einheitsnormale mittels Primärmethoden, Zertifizierte Referenzmaterialien (ZRM) und Referenzverfahren gewährleistet werden. Die quantitative hochauflösende 1H-SP-NMR bietet sich aufgrund ihrer ausgezeichneten Selektivität und ihrem Potenzial als relative Primärmethode geradezu als Referenzverfahren an. Für vier wichtige Anwendungsbereiche (Bestimmung von Stoffmengenverhältnissen und -anteilen in mol/mol bzw. mol/mol %, der Reinheitsbestimmung über die Hauptkomponentenanalyse in g/g % und der Gehaltsbestimmung in mg/g) wurden anhand idealer Modellsysteme in Lösung (5-Komponenten: Ethyl-4-toluolsulfonat, [2,2]-Paracyclophan, Durol, Cyclododekan, Oktamethylcyclotetrasiloxan; Maleinsäure; 3-Trimethyl-2,2,3,3-tetradeuteropropionsäure-Natriumsalz (TSP)) die Messgleichungen und vollständigen Unsicherheitsbudgets aufgestellt sowie Arbeitsanweisungen zur quantitativen Aufnahme und Auswertung von 1H-NMR-Messungen erarbeitet. Dazu war für die Reinheits- und Gehaltsbestimmung ein System interner NMR-Standards aufzubauen (ZRM Benzoesäure, Maleinsäure, TSP-Lösung, Durol), das den Forderungen nach metrologischer Rückführung genügte. Zur Minimierung der Messunsicherheit wurden systematisch die Einflüsse gerätespezifischer Parameter und der Auswertung umfangreich untersucht und quantifiziert. Mittels mitorganisierter nationaler und internationaler CCQM-Ringversuche konnten allgemeingültige (unabhängig von der Gerätekonfiguration) Aussagen über die Messunsicherheit der Methoden bzw. Verfahren getroffen werden. Für realitätsbezogene Fragestellungen der Reinheitsbestimmung möglicher Referenzmaterialien für den pharmazeutischen Bereich (Spiraeosid, Thymol, Loganin) sowie von Xylol-Isomeren-Gemischen und der Gehaltsbestimmung 0,1%-iger wässriger Ethanollösungen mussten teilweise die quantitative 1H-entkoppelte 13C-NMR validiert und der quantitative Einsatz der 1H-Wasserunterdrückung (Presaturation) erstmalig entwickelt werden. Um die Güte der quantitativen NMR-Verfahren als Referenzverfahren bewertet zu können, wurde durch Beteiligung an internationalen Ringversuchen auf höchstem metrologischen Niveau (CCQM) deren Messunsicherheiten mit denen anderer analytischer Verfahren verglichen. Es konnten somit vier Referenzverfahren durch Dokumentation der Prüfbereiche, Messunsicherheiten und Einsatzgebiete der quantitativen hochauflösenden 1H- und 13C-NMR formuliert werden. / The globalisation of trade and economics makes requires mutual international recognition of analytical measurement results. The trueness of analytical results can be secured by establishing traceability to measurement standards for SI units using primary methods, certified reference materials (CRM) and reference methods. The quantitative high resolution 1H-SP-NMR offers itself as reference method due to its excellent selectivity and its potential as relative primary method. For four important areas of application (determination of amount-of-substance ratios and fractions in mol/mol and mol/mol %, respectively, purity determination by main component analysis in g/g %, and determination of minor component mass fractions in mg/g) the measuring equations and complete uncertainty budgets were set up, and work instructions for the acquisition and evaluation of quantitative 1H-NMR measurements were compiled, on the basis of ideal model systems in solution (5 components: Ethyl-4-toluenesulfonate, [2,2]-Paracyclophane, Durene, Cyclododecane, Octamethylcyclotetrasiloxane; Maleic acid; 3-Trimethyl-2,2,3,3-tetradeuteropropionic acid sodium salt (TSP)). In addition, a system of internal NMR standards had to be built up (CRM benzoic acid, maleic acid, TSP solution, Durene) for the determination of composition and purity, which meets the demands for metrological traceability. For the minimization of measurement uncertainty, the influences of instrument-specific parameters and data evaluation techniques were extensively examined and quantified. By means of national and jointly organized international CCQM intercomparisons generally applicable statements (independent of the measuring system configuration) about the measurement uncertainty for the different methods could be specified. For addressing real-life problems in purity determination of prospective reference substances for the pharmaceutical field (spiraeoside, thymol, loganin) as well as of xylene isomer mixtures and the analysis of 0,1 % aqueous ethanol solutions, the quantitative 1H-decoupled 13C-NMR had to be validated in part and the quantitative application of the 1H water suppression (presaturation) was developed for the first time. In order to estimate the power of quantitative NMR as a reference method, measurement uncertainties were compared with those of other analytical methods by participation in international intercomparisons on the highest metrological level (CCQM). Thus, four reference methods of the quantitative high resolution 1H- and 13C-NMR could be specified in terms of measuring ranges, measurement uncertainties and application fields.

Les études non destructives des processus physiologiques dans les produits agronomiques exigent des résolutions spatiales et temporelles de plus en plus élevées. L'imagerie par résonance magnétique nucléaire (RMN) est une technique totalement non-invasive qui permet d'accéder à plusieurs types de variables (architecture des tissus, variabilités spatiales de la composition, flux entrants et internes au cours de la croissance du fruit) plus difficilement quantifiables avec des méthodes destructives classiques. Un des enjeux majeur également réside dans la faculté de localiser spatialement ces transformations physiologiques et morphologiques dans les produits agronomiques. Les travaux de recherches réalisés dans le cadre de cette thèse ont pour objectif principal, la mise en œuvre d'une méthodologie de calcul et d'analyse quantitative en imagerie RMN appliquée à l'agronomie. L'implémentation, l'optimisation et la validation de la séquence FLASH combinée avec des agents de contraste efficaces en terme de relaxivité et bio-compatibles a permis d'une part, la cartographie des paramètres de relaxation et d'autre part, la quantification du transport de l'eau in vivo d'un système agronomique modèle au cours de sa croissance. Les nanoparticules de l'agent de contraste Gd3+[Fe(CN)6]3-/Mannitol ont été utilisées comme des marqueurs afin de localiser les flux hydriques dans le fruit. Le choix de la séquence d'imagerie FLASH a été motivé par la nécessite d'atteindre des résolutions temporelles suffisante pour suivre la dynamique des changements physiologiques liés au transport de l'eau dans ce type de matériau. La validation de la méthode de calcul du T1 menée sur le fantôme a révélé un bon accord par rapport aux T1 mesurés par relaxométrie. Nous avons également mis au point une procédure d'évaluation du rapport signal sur bruit et des incertitudes commises dans chaque voxel des images paramétriques M0 et T1. L'évaluation de ces incertitudes est un élément fondamental de cette analyse quantitative, afin d'assurer des interprétations fiables des images RMN. La segmentation des images nous a permis de localiser précisément les tissus où règne une forte activité cellulaire. Enfin, la modélisation compartimentale mis en oeuvre nous a permis de quantifier les paramètres cinétiques liés au transport de l'eau dans le fruit.Mots-clés: Imagerie RMN quantitative, paramètres intrinsèques, segmentation, modélisation compartimentale, agents de contraste, tissus végétaux / Non destructive studies of physiological processes in agronomic products require increasingly higher spatial and temporal resolutions. Nuclear Magnetic Resonance (NMR) imaging is a completely non-invasive technique providing access to several types of variables (tissue architecture, spatial variability of the composition, external and internal flow during fruit growth) more difficult to quantify with conventional destructive methods. One of major challenge lies in the ability to spatially localize the physiological and morphological changes in the agricultural products. The main objective of the research work in this thesis is to carry out a methodology in order to calculate and analyze quantitative NMR imaging applied to agronomy. The implementation, optimization and validation of the FLASH imaging sequence is performed in combination with innovative biocompatible contrast agents efficient in terms of relativity which allow to map in vivo relaxation parameters and then to explore water transportation in an agronomic model : the tomato during its growth. Nanoparticles of Gd3+[Fe(CN)6]3-/Mannitol contrast agents have been used as markers to localize the water flow in the fruit. The choice of the FLASH imaging sequence is motivated by the necessity to achieve sufficient high temporal resolution for monitoring the dynamics of physiological changes related to the water transport. The validation of the T1 calculation method performed on a phantom shows a good agreement compared to T1 measured by relaxometry. A systematic procedure for the estimation of the signal to noise ratio on the parametric images is also proposed which ensures a carefull determination of the intrinsic parameters of living tissues (M0 and T1) and their uncertainties. This step in the analysis ensures reliable interpretation of NMR images and permits image segmentation in order to precisely localize the tissues where there is a high cellular activity. Finally, the time dependance and the compartmental modeling allow to quantify the kinetic parameters associated with the water transport in the fruit.Keywords: Quantitative NMR imaging, intrinsic parameters, segmentation, compartmental modeling, contrast agents, plant tissues

Une importante proportion de formes atypiques de parkinsonismes a été rapportée en Guadeloupe en 1999. Il ressort des études épidémiologiques menées sur place, que tous les patients atteints étaient de grands consommateurs de produits alimentaires et médicinaux de la famille des Annonaceae, et plus particulièrement du genre Annona. De nombreux genres appartenant à cette famille renferment des molécules fortement cytotoxiques : les acétogénines d’Annonaceae. Leur présence dans les fruits d’Annona muricata a déjà été mise en évidence. Cependant, peu de données existent sur leur teneur en acétogénines dans ces fruits ou dans ceux d’espèces proches. Par ailleurs, bien que l’annonacine, acétogénine principale d’A. muricata soit neurotoxique in vivo, aucune donnée quantitative de son accès au cerveau n’est disponible. Nous nous sommes donc attachés au cours de ce travail, à développer des méthodes de dosage de l’annonacine par LC-MS/MS dans le plasma et le cerveau de Rat pour déterminer sa biodisponibilité et la fraction à tropisme cérébral, dans le but de comprendre pourquoi des molécules si fortement cytotoxiques n’entraînaient pas d’intoxication aigue lors de l’exposition alimentaire. Nous avons par ailleurs développé une méthode de quantification des acétogénines totales par 1H RMN dans des extraits bruts de fruits, appliquée à des lots d’A. muricata d’origines variées. Une méthode par LC-MS/MS a également été développée pour une description plus approfondie des acétogénines présentes dans des extraits bruts de fruits, appliquée à différents lots d’A. squamosa. Les fruits d’A. reticulata et d’A. glabra ont également fait l’objet d’investigations. Ces deux approches combinées ont contribué à améliorer l’estimation de l’exposition aux acétogénines dans le cadre de l’alimentation. / Abstract : A high proportion of atypical parkinsonisms was reported in French West Indies in 1999. Epidemiological studies pointed out an association with the consumption of fruits and medicinal herbs from Annonaceae of the Annona genera. Numerous Annonaceae members contain Annonaceous acetogenins (AAGs), which are highly cytotoxic molecules. They were found in the pulp fruit of Annona muricata. However, scarce only quantitative exist for this fruit and those of related species. Moreover, although annonacin, the major AAG of A. muricata proved neurotoxic in vivo, no quantitative data is available towards its distribution to the brain. We therefore developed a method for annonacin quantitation in Rat plasma and brain homogenate, in order to determine its bioavailability and the fraction reaching the brain, to understand why those highly cytotoxic molecules are not responsible for acute toxicity when fruits are ingested. We then developed a quantitation method for global estimation of AAGs in crude fruit extracts by 1H NMR, which we applied to the fruit pulp of A. muricata batches from diverse locations. An LC-MS/MS method was also developed for the qualitative study of AAGs. It was applied to different batches from A. squamosa fruits. The species A. reticulata and A. glabra were also examined. Those two approaches contributed in a better estimation of AAGs exposure by fruit consumption.