Vernix caseosa is a natural biofilm which covers the skin of a human fetus from the third trimester of pregnancy. It has hydrating, regenerating and anti-microbial effects. The components responsible for these properties of vernix caseosa could be used in the pharmaceutical and cosmetic industries. For this reason, the total composition is analyzed. The lipid components of vernix caseosa consist of squalene, wax esters, sterol esters, 1,2-diol diesters, triacylglycerides, free fatty acids, fatty alcohols, cholesterol, diacylglycerides, monoacylglyceridesů and phospholipids . This study is focused on structure analysis of the 1,2-diol diesters of vernix caseosa. Conditions were optimized for the HPLC separation. Nova -Pak C18 column was used and a gradient of acetonitrile:ethyl acetate was chosen as a mobile phase. Before entering the APCI source ammonium formate was added; ammonium adducts [M +18]+ were formed. Eight scan events was set in MS method. One for the full scan spectrum, second for MS2 spectrum of the precursor ions and six MS3 data-dependent spectra. The position of the double bonds of unsaturated 1,2-diol diesters was determined by fragmentation of their [M+C3H5N]+ adducts. There were identified over 2200 of 1,2-diol diesters differing in lengths of alcohol and fatty acid chains, and...

Les leishmanioses sont des parasitoses en constante évolution, et l’accroissement d’apparitions de résistances vis-à-vis des traitements disponibles en fait une des préoccupations majeures des organismes de santé publique dans le monde. La miltefosine est actuellement le seul antileishmanien actif par voie orale. Son mécanisme d’action implique les lipides et notamment les phospholipides membranaires du parasite.Afin d’évaluer les effets de la miltefosine sur le parasite ainsi que les mécanismes de résistances, une étude lipidomique d’un clone de Leishmania donovani cultivé sous différentes conditions (traité, résistant, résistant-traité) a été réalisée dans le présent travail. Des analyses couplant une séparation des phospholipides en Chromatographie Liquide Haute-Performance à polarité de Phase Normale (NP-HPLC) avec un Spectromètre de Masse (MS) équippé d’une source d’Ionisation ElectroSpray (ESI) ont été traitées par chimiométrie, à l’aide d’une Correction Orthogonale du Signal suivie d’une Analyse Discriminante par Moindre Carrés Partiels (OSC-PLS-DA). Les principales espèces moléculaires permettant de distinguer les différentes cultures ont ensuite fait l’objet d’une identification structurale par spectrométrie de masse en tandem. Des hypothèses métaboliques ont pu être posées.Puis l’étude a été étendue à une plus grande variété de lipides, séparés par NP-HPLC. Pour cela une comparaison des sources d’ionisation à pression atmosphérique (ESI, Ionisation Chimique à Pression Atmosphérique et PhotoIonisation à Pression Atmosphérique) a été nécessaire afin de sélectionner la mieux adaptée pour un tel couplage. Les mécanismes d’action de la miltefosine et de l’amphotéricine B ont alors fait l’objet d’une étude lipidomique. / Leishmaniasis is a more and more spreading disease, and resistance of parasites toward antileishmanial drugs is a concern for public safety organizations troughout the world. Miltefosine is the only oral drug, and its mechanism of action implies membrane lipids, and phospholipids, of parasite cells.In order to assess this mechanism of action, and resistance mechanisms developed, a lipidomic study of Leishmania donovani strains (treated, resistant, treated-resistant) was performed in the present work. A Normal-Phase High-Performance Liquid Chromatography (NP-HPLC) was coupled to an ElectroSpray Ionisation Mass Spectrometer (ESI-MS) to analyze phospholipids, and data were computed using an Orthogonal Signal Correction-Partial Least Squares-Discriminant Analysis (OSC-PLS-DA). Molecular species responsible for the differenciation of strains were then structuraly identified using tandem mass spectrometry. Hypotheses on metabolic pathways implied were then proposed.The study was then extended to a broader range of lipids, also analyzed through NP-HPLC-MS. A comparison of Atmospheric Pressure Ion sources (ESI, Atmospheric Pressure Chemical Ionization and Atmospheric Pressure PhotoIonization) was thus necessary in order to select the most suitable source. A lipidomic study was then performed to assess mechanisms of action and mechanisms of resistance concerning miltefosine and Amphotericin B.

The goal of this project was to develop a rapid separation and detection method for analyzing organic compounds in smokeless powders and then test its applicability on gunshot residue (GSR) samples. In this project, a total of 20 common smokeless powder additives and their decomposition products were separated by ultra performance liquid chromatography (UPLC) and confirmed by tandem mass spectrometry (MS/MS) using multiple reaction monitoring mode (MRM). Some of the targeted compounds included diphenylamines, centralites, nitrotoluenes, nitroglycerin, and various phthalates. The compounds were ionized in the MS source using simultaneous positive and negative electrospray ionization (ESI) with negative atmospheric pressure chemical ionization (APCI) in order to detect all compounds in a single analysis. The developed UPLC/MS/MS method was applied to commercially available smokeless powders and gunshot residue samples recovered from the hands of shooters, spent cartridges, and smokeless powder retrieved from unfired cartridges. Distinct compositions were identified for smokeless powders from different manufacturers and from separate manufacturing lots. The procedure also produced specific chemical profiles when tested on gunshot residues from different manufacturers. Overall, this thesis represents the development of a rapid and reproducible procedure capable of simultaneously detecting the widest possible range of components present in organic gunshot residue.

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Inborn errors of metabolism (IEM) cause significant morbidity and mortality when left untreated. Urine organic acid (UOA) analysis is often a first-line investigation when an IEM is suspected. UOAs are usually qualitatively analyzed via the current gold standard, GC-EI-MS (Gas Chromatography-Electron Impact-Mass Spectroscopy). The Agilent 7890 GC in tandem with the Waters’ Xevo TQ-S MS contains an easily interchangeable LC-ESI (liquid chromatography-electrospray Ionization) and GC-APCI (Atmospheric Pressure Chemical Ionization) instrument set-up, while maintaining accuracy and sensitivity in both LC and GC applications. Utilizing this novel GC-APCI instrument, this project aims to develop and validate a new UOA method for clinical use. Furthermore, utilizing the machine’s MRM mode would increase sensitivities thus allowing for hopefully quantitative analysis. Chemical standards and patient urine samples were extracted via a liquid-liquid ether extraction and derivatized with BSTFA for proper GC elution. Results were compared on the current gold standard GC-EI-MS instrument and the new GC-APCI-MS instrument. Initial instrument suitability and method setup was then optimized. Source moisture levels were modified to explore the wet proton transfer and the dry charge transfer mechanism using [M+H]+ and [M+*]+ ion peak ratios, respectively. Elution times and APCI ion mass spectra profiles of UOA metabolites of interest were identified from full scan mode in preparation for MRM mode analysis. Exploration into the wet and dry mode settings of the APCI source determined that the former induced via methanol had greater peak areas and signal-to-noise ratios. Suitable MRMs were determined for clinically relevant organic acids from which a quantitative assay was developed for methyl malonic acid and several other compounds. The Waters’ Xevo TQ-S micro with Agilent 7890 GC demonstrated promising GC-APCI-MS detection of urine organic acids. With clear avenues for future work, the APCI technique hints at great benefits for biochemical genetic laboratories. / Thesis / Master of Science (MSc) / Inborn errors of metabolism (IEM) are a class of genetic diseases that when left untreated, cause reduced quality of life and sometimes death in newborns. Urine organic acid (UOA) analysis is used for detection using an instrument called GC-EI-MS (Gas Chromatography Electron Impact Mass Spectroscopy). This project explores how a new instrument, the Agilent 7890 GC and the Waters’ Xevo TQ-S MS, can detect these genetic diseases using a technique called APCI (Atmospheric Pressure Chemical Ionization) while still being accurate and sensitive. UOAs are isolated from urine and run through the new machine. When compared to the currently used technique, results were promising but further optimization is needed. Using the new machine, various UOA compounds that were elevated and/or decreased in newborns with genetics diseases were identified and quantified. With clear avenues for future work, the APCI technique can greatly improve newborn diagnosis of IEMs.

The objective of this research was to develop new sample preparation procedures for the isolation of sulfonamides, as well as, to determine the applicability of employing on-line nitrogen selective and mass spectrometric detection methods. The first phase of this research investigated the effect of temperature and pressure on the supercritical fluid extraction (SFE) of sulfonamides from a spiked sand matrix. Temperature effects were either positive or negative with respect to extraction rate and total recovery, depending on the pressure and extraction fluid employed. The second portion of this research compared trifluoromethane (CHF3) and carbon dioxide (CO2) as fluids for the extraction of sulfonamides from spiked non-fat dry milk, beef liver, and egg yolk were found to be more selective using CHF3 than CO2. The polar trifluoromethane improved the extraction efficiency of the polar sulfonamides from the biological matrices and also reduced the amount of co-extractives. The next phase of this research considered the effect of organic modifier and CO2 in the SFE of sulfonamides from chicken liver, beef liver and egg yolk. Methanol, ethanol, acetone, acetonitrile were compared to determine optimum conditions. A SFE method employing 20% acetonitrile modified CO2 yielded quantitative recovery of sulfonamides from chicken liver, but 20% acetone modified CO2 was required to obtain quantitative recovery from beef liver. Either 20% acetone or 20% acetonitrile yielded quantitative recovery from egg yolk. The last phase of this research focused on the evaluation of selective detection methods for sulfonamide analysis. Chemiluminescence nitrogen detection (CLND) parameters were optimized for use with packed column supercritical fluid chromatography (SFC) yielding a minimum detectable quantity (MDQ) of 5 ng of sulfamethazine, on column. Improvements in the detector design decreased the MDQ to 0.5 ng, while, decreasing the column diameter further reduced the MDQ to 125 pg. The second part of this phase evaluated PLC/Atmospheric pressure chemical ionization (APCI) mass spectrometry for the detection of sulfonamides. Sensitivity in selective ion mode was found to be as low as 50 pg on column for sulfamethazine. Supercritical fluid extracts of sulfonamides spiked at 100μg/kg in chicken liver were found to be readily detected by this method. / Ph. D.

<p>Crude oil is a mixture of hydrocarbons so complex that it is predicted to comprise as many compounds as there are genes in the human genome. Developing methods to not only recover crude oil from the ground but also to convert crude oil into desirable products is challenging due to its complex nature. Thus, the petroleum industry relies heavily on analytical techniques to characterize the oil in reservoirs prior to enhanced oil recovery efforts and to evaluate the chemical compositions of their crude oil based products. Mass spectrometry (MS) is the only analytical technique that has the potential to provide elemental composition as well as structural information for the individual compounds that comprise petroleum samples. The continuous development of ionization techniques and mass analyzers, and other instrumentation advances, have primed mass spectrometry as the go-to analytical technique for providing solutions to problems faced by the petroleum industry. The research discussed in this dissertation can be divided into three parts: developing novel mass spectrometry-based methods to characterize mixtures of saturated hydrocarbons in petroleum products (Chapters 3 and 5), exploring the cause of fragmentation of saturated hydrocarbons upon atmospheric pressure chemical ionization to improve the analysis of samples containing these compounds (Chapter 4), and developing a better understanding of the chemical composition of crude oil that tightly binds to reservoir surfaces to improve chemically enhanced oil recovery (Chapter 6). </p>

Lipids from the fat body of queens of Bombus terrestris species in different life stages were studied using chromatographic methods. High-performance liquid chromatography - mass spectrometry with electrospray ionization was used to analyze all lipids from the tissue. Semi- preparative thin-layer chromatography was used to isolate triacylglycerols (TGs) from the fat body tissue. The TGs were subsequently analyzed by high-performance liquid chromatography - atmospheric pressure chemical ionization mass spectrometry. Quantitative differences between different life stages have been found. Qualitative composition has not been changing significantly. Only minor differences have been found in the substances, which were present in amount less than 1%.

<p dir="ltr">Tandem mass spectrometry (MSⁿ) has become the most widely used analytical technique for the chemical characterization of unknown organic compounds in complex mixtures. It has led to the development of a large number of mass spectrometers with different mass analyzers as well as a wide array of ionization methods. This technique can be coupled with a diverse range of chromatography methods, such as gas chromatography (GC) and high-performance liquid chromatography (HPLC). Some of the primary strengths of MS include its great sensitivity, its versatility to seamlessly integrate with various chromatography techniques and its flexibility in the sense of access to different mass analyzers and different ionization methods. During MS experiments, analytes are evaporated and ionized and the resulting ions are separated based on their mass-to-charge (<i>m/z</i>) ratios and then detected. On the other hand, MSⁿ experiments involve isolating a specific ion of interest from all other ions and subjecting them to reactions such as collision-activated dissociation (CAD) or ion-molecule reactions. These reactions generate product ions that can be used to obtain structural information for the analyte. In addition, MSⁿ experiments can be used to generate and study the chemical properties of reaction intermediates, such as oxenium cations. </p><p dir="ltr">The mass spectrometer and the ionization source used to perform the research discussed in this thesis are described in Chapter 2. After this, the development of experiments involving ion-molecule reactions accompanied by collision-activated dissociation in a linear quadrupole ion trap is discussed, with the goals of differentiating the aziridine functionality from structurally related functional groups, such as the amino group and identifying aromatic aldehyde functionalities in protonated oxygen-containing monofunctional analytes. The integration of machine learning with mass spectral data has become an increasingly prevalent and valuable way to interpret data faster and more accurately without human bias than conventional manual approaches. Chapter 5 discusses combining machine learning-guided automated HPLC analysis coupled with MSⁿ experiments based on diagnostic ion-molecule reactions for the structural elucidation of unknown compounds. Finally, experimental and computational studies on the gas-phase reactivity of quinoline-based ground-state singlet oxenium cations are discussed.</p>