<p>The comprehensive analysis of human blood plasma metabolome has been completed using derivatization gas chromatography-mass spectrometry (GC-MS) analysis, liquid chromatography-mass spectrometry (LC-MS) analysis and a comprehensive LC-GC-MS analysis approach wherein LC fractions were collected, derivatized and analyzed using GC-MS. In all cases blood plasma samples were deproteinized using solvent precipitation prior to chromatography and MS analysis.</p> <p>In GC-MS analyses, the progress of all derivatization reactions was monitored by adding 9-anthracenemethanol and 1,3-diphenylacetone to all reaction mixtures; their conversions to 9-anthracenemethanol trimethylsilyl ether and the oxime derivative of 1,3-diphenylacetone were used as measures of the completion of these derivatization reactions. Any reactions with completions less than 99% were repeated.</p> <p>GC-MS analysis of blood plasma samples detected 100 peaks; 44 were positively identified by comparing retention indices and mass spectra with those of authentic standards. LC-MS analyses were conducted on a HILIC column (aminopropyl phase) with MS detection in both negative ion and positive ion modes and resulted in the identification of 97 peaks; 47 were observed in the positive ion mode, 58 in the negative ion mode with 8 peaks observed in both modes.</p> <p>The multi-dimensional LC-GC approach was not designed as a routine analytical method; rather the purpose of this approach was to see how many compounds could be observed in the sample and to obtain better quality mass spectra and retention index values. The LC separation afforded 16 fractions which upon derivatization GC-MS analysis gave an additional 176 peaks from a total of 276 peaks. The MS data from these additional spectra can be used to develop selected ion monitoring GC-MS or tandem mass spectrometry analytical methods. This thesis has demonstrated the power of off-line comprehensive methods to identify compounds that neither the GC nor the LC methods detected.</p> / Master of Science (MSc)

Petit Manseng grapes harvested in 2011 and 2012 were fertilized with soil nitrogen at 0, and 30 kgN/ha, foliar nitrogen at 15kg/ha and foliar nitrogen plus sulfur at 15kg/ha and 5kg respectively. Point quadrat analysis demonstrated foliar nitrogen alone and nitrogen plus sulfur treatments increased percent gaps and lower leaf layer numbers. Berry juice samples differed in ammonia, arginine and yeast assimilable nitrogen concentration. Total glycosides were 25 percent higher in the foliar nitrogen treatment versus the control treatment. Electronic nose measurements on field clusters and laboratory berry analyses was different among treatments in volatile content. Harvest samples underwent acid or enzyme hydrolysis of precursor fractions. Solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC/MS) analysis identified 27 free aroma and flavor compounds and 52 bound compounds. Lactones and carboxylic acids were the major components of the free fractions while bound fractions had increased concentrations of alcohols, esters and terpenes compared to the free fraction. With nitrogen fertilization, acid and enzyme hydrolysis had reduced concentrations of some higher alcohols and carboxylic acids. Acid hydrolysis released more terpenes with nitrogen treatments versus enzymatic hydrolysis. Ester content was increased in both acid and enzyme hydrolysis fractions in vines receiving nitrogen treatments. For descriptive analysis, eight trained panelists described aroma, flavor, texture/mouthfeel and aftertaste attributes. Analysis of Variance (ANOVA) demonstrated that wines were a significant source of variation with 23 of the 24 attributes used. Wine principal component analysis (PCA) of aroma attributes explained 23.5% of the variation from PC1, while flavor-by-mouth and texture/mouthfeel attributes explained 26.3% of the variation due to PC1. The aim of this study was to develop descriptive terms for Petit Manseng and determine the influence of fruit nitrogen levels on the aroma and flavor profile of this cultivar. / Ph. D.

Ethoxyquin is an antioxidant commonly used to preserve vitamins and lipids in various food products and animal feeds. The extraction and determination of ethoxyquin is becoming increasingly important as products, which are labeled as "natural" are becoming more common. The present method of determination only ensures that ethoxyquin values are below 10-20 parts per million. Therefore, advances are needed in methods of extraction and analysis in order to lower the detection limits in various products. The first part of this research investigates the use of supercritical fluids in the extraction of ethoxyquin from lean beef and beef fat. Supercritical fluids offer the advantages of safety, time, expense, and selectivity over liquid extractions. Three fluids were examined: carbon dioxide, trifluoromethane, and 1,1,1,2-tetrafluoroethane. Carbon dioxide appeared to react with ethoxyquin during the extraction. Methanol modified hydrofluorocarbons provided more complete extractions over pure hydrofluorocarbon fluids. Methanol modified 1,1,1,2-tetrafluoroethane was used in the extraction of ethoxyquin from lean beef and beef fat, and provided a quantitative extraction at the 0.5 ppm level. The second part of this research centered on the separation and quantitation of the oxidation products of ethoxyquin through the use of high pressure liquid chromatography with chemiluminescence nitrogen detection (HPLC/CLND). When ethoxyquin is oxidized, the resulting products also exhibit antioxidative properties. While these oxidation products are known, no effort has been made to separate and quantify them in real or clean samples. HPLC/CLND allows all nitrogen containing compounds to be quantified without a known standard. This method is of extreme interest in the case of ethoxyquin oxidation products, or other types of metabolites, where standards are difficult to obtain or are unstable. HPLC/CLND allowed a separation of ethoxyquin and four of its oxidation products to be detected, thus making future studies of the antioxidant behavior of ethoxyquin feasible. / Master of Science

An alternative method for extraction of therapeutically beneficial compounds such as sterols, fatty acids, and tocopherols from cranberry seeds with pure SF CO2 has been provided. The supercritical fluid extraction (SFE) operating conditions such as extraction temperature, pressure of CO2, extraction time, and CO2 flow rate were optimized to maximize the extraction yield. The amount and type of SF extractables (pure CO₂) have been compared with Soxhlet extractables (hexane) to evaluate the feasibility of SFE as an alternative extraction method. The extractables obtained via hexane and SF CO₂, which were derivatized and identified by gas chromatography mass spectrometry (GC-MS), contained mostly methylated fatty acids. / Master of Science

Tesis por compendio / [EN] Fruits both produce and emit volatile chemical compounds. These are short-chained low polarity molecules involved in many processes, and they are responsible of our perception of fruit aroma and of most of their flavour. This thesis is focused on the study of volatile compounds in the tomato fruit, which is one of the most important horticultural worldwide and is a model system for the study of fruit development and ripening. Some of the analytical methods more frequently used for the analysis of tomato fruit volatiles were systematically compared. Results revealed that the observed volatile profile is highly dependent on the precise analytical method used, both for sample processing and for the technique used for volatile acquisition. It was concluded that the method of election for the comparison of large sets of samples from a multi-omics approach consists on flash freezing the biological material with liquid nitrogen at the selected ripening stage and the use of headspace solid phase microextraction coupled to gas chromatography and mass spectrometry for the subsequent analysis. This method was implemented and was used for the determination of volatile compounds in selected NILs harbouring QTLs for characters related to flavour and aroma introgressed in different genetic backgrounds. The results allowed the association of several of the organoleptic characters previously identified with modified levels of several volatiles. It was also observed that the genetic background has a major effect on the production of such metabolites. Correlation analysis between the levels of volatiles and primary metabolites led to the conclusion that the production of volatile compounds is generally not determined by the levels of their precursors. Its regulation is most likely to be due to downstream processes such as the availability of either precursors or intermediate metabolites, the variability in specific processes leading to the concersion of precursors into volatiles, or to other still unknown regulatory mechanisms. Volatile compounds were also studied in a RIL population derived from a cross between Solanum pimpinellifolium accession TO-937, the closest species to cultivated, which produces red fruits, and S. lycopersicum cv. 'Moneymaker', a tomato variety for the fresh market. This allowed the identification of 102 QTLs for 39 different volatile compounds, 76 of which had not been previously described. All these QTLs were mapped along the 12 tomato chromosomes by means of the SOLCAP SNPs molecular market map. Most of the QTLs identified were subsequently evaluated on introgression lines (ILs) generated from the same original genotypes. It was observed that almost half of the QTLs previously identified retained their effect after introgression in the 'Moneymaker' genetic background. Additionally, 12 new QTLs were identified in this IL population. Based on the existing knowledge about the effect of volatile compounds on our perception of flavour and aroma and also on their ability to maintain their effect after introgression in the cultivated tomato, some of the QTLs identified are good candidates to be used in tomato flavour breeding programs. Eventually, the comparison of the localization in the genome of the QTLs identified in the different populations studied with those already described in the literature revealed a very low degree of co-localization between the different QTLs. This implies that there exists a wide range of variability in the wild species related to tomato available for breeding tomato flavour and aroma. / [ES] Los frutos producen y emiten compuestos químicos volátiles. Estos son moléculas en general poco polares y de cadena corta que cumplen diversas funciones, y son las responsables de que percibamos el aroma y buena parte del sabor de los frutos. Esta tesis está centrada en el estudio de los volátiles del fruto del tomate, que es uno de los cultivos hortícolas más importantes y un sistema modelo para el estudio del desarrollo y maduración del fruto. Se compararon de forma sistemática los métodos analíticos más comúnmente utilizados para el análisis de volátiles en fruto de tomate, y se observó que el perfil de volátiles detectado está fuertemente condicionado por el método analítico utilizado, tanto por el proceso de preparación de la muestra como por la técnica de adquisición de los volátiles. Finalmente se concluyó que la técnica más adecuada para la comparación de grandes grupos de muestras desde una aproximación multi-ómica consiste en congelar con nitrógeno líquido el material vegetal una vez alcanzado el momento idóneo de recolección, y su análisis posterior mediante microextracción en fase sólida (SPME) acoplada a cromatografía de gases y espectrometría de masas. Se puso a punto esta técnica y se utilizó para la determinación de los compuestos volátiles en varias líneas NILs portadoras de QTLs de caracteres relacionados con el sabor y el aroma en distintos fondos genéticos. Los resultados permitieron asociar varios de los caracteres organolépticos identificados con alteraciones en los niveles de algunos volátiles. Igualmente se observó que el fondo genético tiene un efecto importante sobre la producción de estos metabolitos. Los análisis de correlación entre los niveles de volátiles y metabolitos primarios permitieron concluir que la producción de compuestos volátiles, en general, no está determinada por los niveles de sus precursores, sino que su regulación debe encontrarse más bien en procesos posteriores, tales como la disponibilidad de los precursores o de metabolitos intermedios, variabilidad en procesos específicos relacionados con la conversión de los precursores en volátiles, o algún otro mecanismo regulador aún desconocido. También se estudiaron los volátiles en una población de RILs derivada de un cruce entre Solanum pimpinellifolium entrada TO-937, la especie más próxima al tomate cultivado, la cual produce frutos rojos, y S. lycopersicum cv. "Moneymaker", una variedad de tomate para el mercado en fresco. Esto permitió identificar 102 QTLs para 39 volátiles diferentes, 76 de las cuales no se habían descrito previamente, las cuales se mapearon a lo largo de los 12 cromosomas del tomate utilizando el mapa de marcadores moleculares de SNPs SOLCAP. Posteriormente se evaluaron la mayoría de las QTLs identificadas mediante la determinación de los volátiles en líneas ILs generadas a partir de los mismos materiales. Se observó que casi la mitad de estas QTLs mantuvieron su efecto al ser introgresadas en el fondo genético "Moneymaker", al tiempo que 12 nuevas QTLs se identificaron en esta población de ILs. Algunas de las QTLs identificadas, en base al conocimiento existente sobre el efecto de los compuestos volátiles en nuestra percepción del sabor y el aroma, y en base a su capacidad para mantener su efecto tras su introgresión en el tomate cultivado, resultan ser candidatos prometedores para su utilización en la mejora genética del sabor del tomate. Finalmente, el análisis de la localización en el genoma de las QTLs analizadas en las distintas poblaciones objeto de estudio en esta tesis, junto con las descritas en la bibliografía disponible, puso de relieve el bajo grado de co-localización existente entre las distintas QTLs, lo cual implica que en las especies silvestres relacionadas con el tomate existe un amplio rango de variabilidad genética susceptible de ser utilizado para la mejora de su sabor y su aroma. / [CA] Els fruits produixen i emitixen compostos químics volàtils. Estos són molècules en general de baixa polarita i cadena curta que tenen diverses funcions, i són les responsables de la nostra percepció de l'aroma i de bona part del sabor dels fruits. Esta tesi està centrada a l'estudi dels volàtils del fruit de la tomata, que és un dels cultius hortícoles més importants i un sistema model per a l'estudi del desenvolupament i la maduració del fruit. Es van comparar de forma sistemática els mètodes analítics més habituals per a l'anàlisi de volàtils en fruits de tomata, i es va observar que el perfil de volàtils detectat està fortament condicionat per el mètode analític utilitzat, tant per el procés de preparació de la mostra com per la técnica d'adquisició dels volàtils. Es va concluir que la t`cnica més adequada per a la comparació de grans grups de mostres desde una aproximació multi-òmica consistix en congelar en nitrògen líquid el material vegetal en el momento idoni de recolecció, i analitzar-lo posteriorment per microextracció en fase sòlida (SPME) acoplada a cromatografía de gasos i espectrometría de masses. Es va posar a punt esta técnica i es va utilitzar per a la determinació dels composteos volàtils en varies línies NILs portadores de QTLs de caràcters relacionats en el sabor i l'aroma en fons genètics diversos. Els resultats van permetre associar alguns dels caràcters organolèptics identificats en alteracions en els nivells d'alguns compostos volàtils. També es va observar que el fons genètic té un efecte important sobre la producción d'estos metabolits. Els anàlisi de correlació entre els nivells de volàtils i els metabolits primaris ens van permetre concluir que la producción de compostos volàtils, en general, no està determinada per els nivells dels seus precursors. La seua regulació és deguda a procesos posteriors, com la disponibilitat dels precursors o de metabolits intermediaris, la variabilitat en processos específics relacionats en la conversió dels precursors en volàtils, o en algún altre mecanisme regulador encara desconegut. També es van estudiar els volàtils en una población de RILs rerivada d'un creuament entre Solanum pimpinellifolium entrada TO-937, la espècie silvestre més próxima a la tomatera cultivada i que produix fruits rojos, i S. lycopersicum cv. Moneymaker, una varietat de tomata per al mercat en fresc. Açò va permetre l'identificació de 102 QTLs de 39 volàtils diferents, 76 de les quals no s'havien descrit prèviament, i que es van mapejar al llarg dels 12 cromosomes de la tomatera mitjançant el mapa de marcadors moleculars de SNPs SOLCAP. Posteriorment es van evaluar la majoria de les QTLs identificades mitjançant la determinació dels volàtils en línies ILs generades a partir dels mateixos materials. Es va observar que quasi la meitat de estes QTLs van mantindre el seu efecte al ser introgressades en el fons genètic "Moneymaker". Adicionalment, 12 noves QTLs es van identificar en esta población d'ILs. Algunes de les QTLs identificades, en base al coneiximent existent respecte a l'efecte dels volàtils en la nostra percepció del sabor i l'aroma, i tenin en cónter la seua capacitat de mantindre el seu efecte al ser introgressats en la tomata cultivada, són candidats prometedors per a ser utilitzats en la millora genética del sabor de les tomates. Finalment, l'anàlisi de la localització en el genoma de les QTLs analitzades en les distintes poblacions objecte d'aquesta tesi, junt a les descrites en la bibliografía disponible, va evidenciar que existix una baixa freqüència de co-localització entre les distintes QTLs. Açò implica que existix molta variabilitat genética en les espècies silvestres relacionades en la tomatera, que pot ser utilitzada per a la millora del sabor i l'aroma dels seus fruits. / Rambla Nebot, JL. (2016). GENES AND GENOMIC REGIONS RELATED TO THE PRODUCTION OF VOLATILE COMPOUNDS IN THE TOMATO FRUIT [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61768 / Premios Extraordinarios de tesis doctorales / Compendio

Identification of disease-related alterations in molecular and cellular mechanisms may reveal useful biomarkers for human diseases including cancers. High-throughput omic technologies for identifying and quantifying multi-level biological molecules (e.g., proteins, glycans, and metabolites) have facilitated the advances in biological research in recent years. Liquid (or gas) chromatography coupled with mass spectrometry (LC/GC-MS) has become an essential tool in such large-scale omic studies. Appropriate LC/GC-MS data preprocessing pipelines are needed to detect true differences between biological groups. Challenges exist in several aspects of MS data analysis. Specifically for biomarker discovery, one fundamental challenge in quantitation of biomolecules is owing to the heterogeneous nature of human biospecimens. Although this issue has been a subject of discussion in cancer genomic studies, it has not yet been rigorously investigated in mass spectrometry based omic studies. Purification of mass spectometric data is highly desired prior to subsequent differential analysis. In this research dissertation, we majorly target at addressing the purification problem through probabilistic modeling. We propose an intensity-level purification model (IPM) to computationally purify LC/GC-MS based cancerous data in biomarker discovery studies. We further extend IPM to scan-level purification model (SPM) by considering information from extracted ion chromatogram (EIC, scan-level feature). Both IPM and SPM belong to the category of topic modeling approach, which aims to identify the underlying "topics" (sources) and their mixture proportions in composing the heterogeneous data. Additionally, denoise deconvolution model (DMM) is proposed to capture the noise signals in samples based on purified profiles. Variational expectation-maximization (VEM) and Markov chain Monte Carlo (MCMC) methods are used to draw inference on the latent variables and estimate the model parameters. Before we come to purification, other research topics in related to mass spectrometric data analysis for cancer biomarker discovery are also investigated in this dissertation. Chapter 3 discusses the developed methods in the differential analysis of LC/GC-MS based omic data, specifically for the preprocessing in data of LC-MS profiled glycans. Chapter 4 presents the assumptions and inference details of IPM, SPM, and DDM. A latent Dirichlet allocation (LDA) core is used to model the heterogeneous cancerous data as mixtures of topics consisting of sample-specific pure cancerous source and non-cancerous contaminants. We evaluated the capability of the proposed models in capturing mixture proportions of contaminants and cancer profiles on LC-MS based serum and tissue proteomic and GC-MS based tissue metabolomic datasets acquired from patients with hepatocellular carcinoma (HCC) and liver cirrhosis. Chapter 5 elaborates these applications in cancer biomarker discovery, where typical single omic and integrative analysis of multi-omic studies are included. / Ph. D. / This dissertation documents the methodology and outputs for computational deconvolution of heterogeneous omics data generated from biospecimens of interest. These omics data convey qualitative and quantitative information of biomolecules (e.g., glycans, proteins, metabolites, etc.) which are profiled by instruments named liquid (or gas) chromatography and mass spectrometer (LC/GC-MS). In the scenarios of biomarker discovery, we aim to find out the significant difference on intensities of biomolecules with respect to two specific phenotype groups so that the biomarkers can be used as clinical indicators for early stage diagnose. However, the purity of collected samples constitutes the fundamental challenge to the process of differential analysis. Instead of experimental methods that are costly and time-consuming, we treat the purification task as one of the topic modeling procedures, where we assume each observed biomolecular profile is a mixture of hidden pure source together with unwanted contaminants. The developed models output the estimated mixture proportion as well as the underlying “topics”. With different level’s purification applied, improved discrimination power of candidate biomarkers and more biologically meaningful pathways were discovered in LC/GC-MS based multi-omic studies for liver cancer. This research work originates from a broader scope of probabilistic generative modeling, where rational assumptions are made to characterize the generation process of the observations. Therefore, the developed models in this dissertation have great potential in applications other than heterogeneous data purification discussed in this dissertation. A good example is to uncover the relationship of human gut microbiome with the host’s phenotypes of interest (e.g., disease like type-II diabetes). Similar challenges exist in how to infer the underlying intestinal flora distribution and estimate their mixture proportions. This dissertation also covers topics of related data preprocessing and integration, but with a consistent goal in improving the performance of biomarker discovery. In summary, the research help address sample heterogeneity issue observed in LC/GC-MS based cancer biomarker discovery studies and shed light on computational deconvolution of the mixtures, which can be generalized to other domains of interest.

No / References Citations Metrics Reprints & Permissions Get access Abstract This paper presents the observation of lipid residue, identified as beeswax, preserved in the ceramic matrix of a Late Chalcolithic (c. 3700–3000 BC) bevelled-rim bowl (BRB) from the site of Tepe Sofalin on the Tehran Plain. Gas chromatography-mass spectrometry (GC-MS) was used to separate and identify the lipid constituents preserved in the matrix of a BRB sherd. Lipid biomarkers were recovered including long-chain n-alkanes, n-alkenes, palmitic wax monoesters, fatty acids and n-alcohols characteristic of beeswax. In addition to two disaccharides, cholesterol and β-sitosterol as contaminants were retrieved by solvent soluble extraction from a number of different locations from the ceramic matrix of the analysed sherd.

Yes / Vast numbers of votive mummies were produced in Egypt during the Late Pharaonic, Ptolemaic, and Roman periods. Although millions remain in situ, many were removed and have ultimately entered museum collections around the world. There they have often languished as uncomfortable reminders of antiquarian practices with little information available to enhance their value as artefacts worthy of conservation or display. A multi-disciplinary research project, based at the University of Manchester, is currently redressing these issues. One recent aspect of this work has been the characterization of natural products employed in the mummification of votive bundles. Using gas chromatography–mass spectrometry and the well-established biomarker approach, analysis of 24 samples from 17 mummy bundles has demonstrated the presence of oils/fats, natural waxes, petroleum products, resinous exudates, and essential oils. These results confirm the range of organic materials employed in embalming and augment our understanding of the treatment of votives. In this first systematic initiative of its kind, initial findings point to possible trends in body treatment practices in relation to chronology, geography, and changes in ideology which will be investigated as the study progresses. Detailed knowledge of the substances used on individual bundles has also served to enhance their value as display items and aid in their conservation. / RCB is supported by a PhD studentship from the Art and Humanities Research Council (43019R00209). L.M. and S.A.W. are supported by a Leverhulme Trust Research Project Award (RPG-2013-143).

No / To analyze the fatty acid contents of Buddleja asiatica Lour, both the non-volatile oil and fat obtained from the n-hexane soluble sub-fraction were subjected to GC/MS using BSTFA (N,O-bis(trimethylsilyl) trifloroacetamide) derivatization. The oil showed the presence of six fatty acids including palmitic acid (46.75 %), linoleic acid (37.80 %), stearic acid (10.98 %), arachidic acid, margaric acid and lignoceric acid (< 3 %). Analysis of the fat revealed nine fatty acids including lignoceric acid (43.12 %), behenic acid (26.39 %), arachidic acid (9.29 %) and stearic acid (5.3 %). Cerotic acid, montanic acid, melissic acid and palmitic acid were found in low amounts (< 5 %) while trycosylic acid (4.83 %) was the only fatty acid with odd number of carbon atoms. The oil showed a low thermal stability. / Higher Education Commission Pakistan

By addressing new sample preparation techniques, the U.S. Environmental Protection Agency (EPA) has recently implemented research programs to reduce or abolish laboratory pollution. In the work reported here, EPA Method 8270, established for Priority Pollutant Organics, will be evaluated by both Soxhlet and microwave-assisted extraction (MAE) for two classifications of compounds. The common procedure for sample preparation of solids is Soxhlet extraction. This is a lengthy operation and uses abundant solvent volumes. With ever changing times, the new technology of MAE is surfacing. This technique uses far less solvent and sample preparation times are greatly reduced. The work reported here compares the recoveries of phenolic and polynuclear aromatic compounds for both Soxhlet and closed-vessel MAE. / Master of Science