Nesta dissertação foram avaliadas metodologias para reação de amidação de amostras comerciais de poli (metilmetacrilato), PMMA, com alilamina e benzilamina. A reação foi executada em tubo selado sob aquecimento a 90 C por sete dias, sem que nenhuma quantidade mensurável de amida pudesse ser detectada por FTIR e RMN. A reação foi reavaliada sob catalise de DBU, 3,4% molar, sem que nenhum resultado positivo obtido. O emprego de DBU associado ao NaCN como co-catalisador mostrou-se eficaz é o PMMA pode ser derivatizado em 30 %, com alilamina, e 13 % com benzilamina, sob as mesmas condições de temperatura e tempo. A analise elementar e o RMN-1H foram conclusivos na quantificação das reações enquanto o FTIR mostrou-se pouco eficaz devido a sobreposições de bandas. A taticidade das amostras comerciais de PMMA e do material produzido pode ser avaliada por RMN. Experimentos exploratórios efetuados em forno de microondas monomodo sob as mesmas relações molares com aquecimento a 120 C por 30 minutos com 80 W de potencia não foram capazes de promover alterações na matriz polimérica / This work evaluated methodologies for amidation reaction of commercial samples of poly (methylmethacrylate), PMMA, with allylamine and benzylamine. The reaction was performed in a tube under heating at 90 C for seven days without any measurable amount of amide could be detected by FTIR and NMR. The reaction was reassessed under catalysis of DBU, 3,4 mol %, without any positive results. The use of DBU associated with NaCN as co-catalyst was effective with PMMA can be derivatized by 30%, with allylamine, and 13% with benzylamine under the same conditions of temperature and time. The elemental analysis and 1H-NMR were conclusive in quantifying the reactions while FTIR was found to be unreliable due to overlapping of bands. The tactic of commercial samples of PMMA and the material produced can be assessed by NMR. Exploratory experiments conducted in single-mode microwave oven under the same molar ratios heated at 120 C for 30 minutes with 80 W of power were not able to promote changes in the polymer matrix

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The study of metabolism through the metabolites determination is assuming great importance in biological scientific scene, once it allows the knowledge or even understanding of how and why they are modified by existing routes in biological matrices. Microalgae are unicellular organisms prokaryotes or eukaryotes with photosynthetic capacity, that presents relatively simple requirements for growth and high potential to technological interest products synthesis. Thus, the aim of this study was to develop a simultaneous extraction method of non-polar fraction (fatty acids) and polar (amino acids and organic acids) in microalgae intracellular content, employing gas chromatography (GC) as analytical analysis tool. The compounds were extracted by Bligh e Dyer method with reducing the volume organic solvents and different forms of agitation and/or cell disruption. The non-polar compounds were derivatizated by Hatman e Lago method. The derivatization of polar compounds was performed according to Oms-oliu and validated for microalgae biomass sample, considering selectivity, linearity, detection and quantification limits, precision and accuracy. Then, were analyzed two chlorophytes, Chlorella vulgaris and Scenedesmus obliquus and two cyanobacteria, Aphanothece microscopica Nägeli and Phormidium autumnale. Eleven fatty acids, ten amino acids and three organic acids were detected between the species. The succinic, malic and citric acids, important intermediates from tricarboxylic acid cycle (TCA), from which are formed microalgae products of interest. Among the amino acids, the glutamic acid was the major compound in all species. Excepting the S. obliquus, the microalgae showed fatty acid profile mostly with saturated carbon chain. Posteriorly, the organic acids, amino acids and fatty acids were characterized during growth of S. obliquus microalgae cultivated photosynthetically with constant lighting. The succinic acid showed constant concentrations after 120 h of cultivation, while malic and citric acids had a significantly higher concentration to 336 h. Excepting the alanine, glutamic acid and asparagine, which showed peak in 336 h, the highest concentrations of amino acids were observed between 120 and 288 h. The fatty acid profile showed significant change between analysis of 0 to 120 h. Firstly the cultivation was characterized by the high concentration of saturated FA and short and medium chain compounds. After 120 h was detected higher concentration of unsaturated AG and long-chain (≥ 20 carbons). In this regard, it was possible establish relationship between microalgae biosynthetic pathways. / O estudo do metabolismo, a partir da determinação de metabólitos, está assumindo elevada importância no cenário científico biológico, pois possibilita o conhecimento ou até mesmo o entendimento de como e porque são modificadas pelo meio as rotas existentes em matrizes biológicas. As microalgas são organismos unicelulares procariontes ou eucariontes com capacidade fotossintética, apresentam requisitos relativamente simples para o crescimento e elevado potencial para síntese de produtos de interesse tecnológico. Dessa forma, o objetivo deste trabalho foi desenvolver um método de extração simultânea da fração apolar (ácidos graxos) e polar (aminoácidos e ácidos orgânicos) do conteúdo intracelular de microalgas, empregando a cromatografia em fase gasosa (GC) como ferramenta analítica de análise. Os compostos foram extraídos pelo método de Bligh e Dyer com redução no volume de solventes orgânicos e diferentes formas de agitação e/ou ruptura celular. Os compostos apolares foram derivatizados segundo Hartman e Lago (1973). A derivatização dos compostos polares foi realizada de acordo com Oms-Oliu et al. (2011) e validada para a amostra biomassa microalgal quanto a seletividade, linearidade, limites de detecção e quantificação, precisão e exatidão. Então, foram analisadas duas clorofíceas, Chlorella vulgaris e Scenedesmus obliquus e duas cianobactérias, Aphanothece microscopica Nägeli e Phormidium autumnale. Foram detectados onze ácidos graxos, dez aminoácidos, e três ácidos orgânicos entre as espécies. Os ácidos succínico, málico e cítrico, importantes intermediários do ciclo do ácido tricarboxílico (TCA), a partir do qual são formados produtos microalgais de interesse. Dentre os aminoácidos, o ácido glutâmico foi o composto majoritário em todas espécies. Com exceção da S. obliquus, as microalgas apresentaram perfil de ácidos graxos (AG) majoritariamente de cadeia carbônica saturada. Posteriormente, foram caracterizados os ácidos orgânicos, os aminoácidos e os ácidos graxos ao longo do crescimento da microalga S. obliquus cultivada fotossinteticamente com iluminação constante. O ácido succínico apresentou concentrações constantes a partir das 120 h de cultivo, enquanto os ácidos málico e cítrico tiveram aumento significativo de concentração às 336 h. Com exceção da alanina, ácido glutâmico e asparagina que apresentaram ápice em 336 h, as maiores concentrações aminoácidos foram observadas entre 120 e 288 h. O perfil de ácidos graxos apresentou mudança significativa entre as análises de 0 e 120 h. Primeiramente o cultivo foi caracterizado pela elevada concentração de AG saturados e compostos de cadeia curta e média, a partir de 120 h foi detectado maior concentração de AG insaturados e de cadeia longa (≥ 20 carbonos). Em face disso, foi possível estabelecer relação entre vias biossintéticas das microalgas.

Mass spectrometry imaging (MSI) has been extensively used to produce qualitative maps of distributions of proteins, peptides, lipids, neurotransmitters, small molecule pharmaceuticals and their metabolites directly in biological tissue sections. Moreover, during the last 10 years, there has been growing demand to quantify target compounds in tissue sections of various organs. This thesis focuses on development and application of a novel instrument- and manufacturer-independent MSI software suite, msIQuant, in the open access format imzML, which has been developed specifically for quantitative analysis of MSI data. The functionality of msIQuant facilitates automatic generation of calibration curves from series of standards that can be used to determine concentrations of specific analytes. In addition, it provides many tools for image visualization, including modules enabling multiple interpolation, low intensity transparency display, and image fusion and sharpening. Moreover, algorithms and advanced data management modules in msIQuant facilitate management of the large datasets generated following rapid recent increases in the mass and spatial resolutions of MSI instruments, by using spectra transposition and data entropy reduction (at four selectable levels: coarse, medium, fine or superfine) before lossless compression of the data. As described in the thesis, implementation of msIQuant has been exemplified in both quantitative (relative or absolute) and qualitative analyses of distributions of neurotransmitters, endogenous substances and pharmaceutical drugs in brain tissue sections. Our laboratory have developed a molecular-specific approach for the simultaneous imaging and quantitation of multiple neurotransmitters, precursors, and metabolites, such as tyrosine, tryptamine, tyramine, phenethylamine, dopamine, 3-methoxytyramine, serotonin, gamma-aminobutyric acid (GABA), and acetylcholine, in histological tissue sections at high spatial resolution by matrix-assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) MSI. Chemical derivatization by charge-tagging primary amines of analytes significantly increased the sensitivity, enabling mapping of neurotransmitters that were not previously detectable by MSI. The two MSI approaches have been used to directly measure changes in neurotransmitter levels in specific brain structures in animal disease models, which facilitates understanding of biochemical mechanisms of drug treatments. In summary, msIQuant software has proven potency (particularly in combination with the reported derivatization technique) for both qualitative and quantitative analyses. Further developments will enable its implementation in multiple operating system platforms and use for statistical analysis.

Les oestrogènes font partie de la famille des neurostéroïdes. Notamment, l’Estradiol (17β-E2) pourrait avoirdes effets bénéfiques sur certaines anomalies liées à l'âge (déclin mnésique et cognitif, maladie d'Alzheimer). Le dosagede très faibles teneurs du 17β-E2 et de ces métabolites (Estrone, Estriol), dans les tissus cérébraux et le plasma, constitueun outil indispensable pour estimer la variation de leurs niveaux avec l'âge et dans différentes pathologies.Tout d'abord, nous avons élaboré une méthode en RP-HPLC-ESI(-)-MS/MS de quantification des oestrogènes sansdérivation chimique. La sélectivité de la séparation et la sensibilité de la détection de ces molécules ont été amélioréesaprès optimisation des paramètres MRM et chromatographiques.Puis, différents agents de dérivation ont été synthétisés et évalués afin d’augmenter le taux d’ionisation pour améliorerla sensibilité de détection. Après l’analyse ESI-MS et MS/MS de onze dérivés du 17β-E2, nous nous sommes intéressésplus particulièrement au dérivé 17β-E2-Q8S.Afin de réduire les interférences, une méthode de préparation de l’échantillon biologique (tissus cérébral, plasma desouris) a été développée. Les deux dosages (sans et avec dérivation) ont été comparées en termes de sensibilité, limitesde détection et de quantification (LD et LQ), linéarité et reproductibilité. Puis, elles ont été appliquées à l’analyse deséchantillons de cerveau, d'hippocampe et de plasma prélevés sur des animaux jeunes et âgés traités avec des quantitésconnues de 17β-E2. La méthode qui nous est apparue la plus robuste est la méthode sans dérivation avec une LD de0,5 fmole.μL-1. / Estrogens are neurosteroids, especially Estradiol (17β-E2) which is considered to be the most biologicallyactive form. 17β-E2 could have positive effects on some age-associated anomalies (memory and cognitive impairment,Alzheimer's disease). For elucidating and better understanding the molecular and cellular mechanisms that underliethe effects within the brain, it is necessary to quantitate 17β-E2 and its metabolites (estrone and estriol) in brain andplasma.First, the RP-HPLC-ESI(-)-MS/MS method without derivatization was developed. The selectivity of the separationand the sensitivity of detection of the estrogens has been improved after optimization of MRM and chromatographicparameters.Secondly, the various derivatization agents were evaluated after their synthesis in order to improve the sensitivity,selectivity and signal enhancement. After studying the eleven synthesized derivatives of 17β-E2 in ESI-MS andMS/MS, promising results were obtained with the 17β-E2-Q8S derivative.A simple purification method using liquid-liquid extraction followed by C18 solid phase extraction has been optimizedin order to minimize assay interferences. The two assays (with and without derivatization) were then compared interms of efficiency, detection and quantification limits (LOD/LOQ), calibration linearity and reproducibility. Then,both methods were validated on biological samples (brain, hippocampus and plasma) collected from animals treatedwith known amounts of 17β-E2. Finally, the more robust method was the method without derivatization with a LODof 0.5 fmol.μL-1.

An efficient two step procedure has been developed using CI GC/MS for analyzing amphetamines and related compounds. The first step allows the analysis of underivatized amphetamines with the necessary sensitivity and specificity to give spectral identification, including differentiation between methamphetamine and phentermine. The second step involves preparing a chiral derivative of the extract to identify d and 1-isomeric composition.

Proteinogenic amino acids are the basic structural building units of proteins. Their analysis is important in many fields, especially in medicine. This thesis deals with one of the methods of derivatization of amino acids in order to increase their absorption in the UV - the post-column derivatization using copper (II) oxide. It is quick, robust and easy-to-use method. Following the nowadays trend in the analysis of free amino acids, this thesis is focused on the HILIC mode separation environment.

In this work, an enantioselective HPLC method with UV and fluorimetric detection was developed and subsequently optimized for chiral separation of four aminoacids (D/L-alanine, D/L-valine, D/L-leucine, D/L-isoleucine) in native and particularly in derivatized form with an emphasis on enantioseparation of D-analogues. Retention and enantioseparation behavior of studied analytes was investigated on three chiral stationary phases based on teicoplanin (Chirobiotic T, Chirobiotic T2) and teicoplanin aglycone (Chirobiotic TAG). At the Chirobiotic T column, enantioseparations of underivatized aminoacids were performed with UV detection at 205 nm in the mobile phases methanol/water with different volume ratios. Baseline separation of L- and D-forms was achieved, however, the sensitivity of detection was very low. In order to increase detection sensitivity, derivatization of aminoacids was performed using 9-fluorenylmethyl chloroformate (FMOC-Cl) and the derivatization procedure was monitored on Chirobiotic T column with fluorimetric detection (λEx = 254 nm, λEm = 314 nm) in a buffered mobile phase methanol/0.5% TEAA buffer, pH 6.0 40/60 (v/v). In terms of derivatization, volume ratio D/L-aminoacid/derivatization agent 1/1 with ten times higher concentration of derivatization agent was found to be the most suitable....

Although Lake Victoria is threatened by pollution there is a lack of knowledge about pharmaceuticals contaminants drained into the lake from large cities bordering the lake. Hence, the purpose of this project was to develop, validate and apply a method for analysis of pharmaceutical compounds accumulating in the Lake Victoria sediments. A simple quantitative method for 11 pharmaceuticals combining accelerated solvent extraction, solid phase extraction, trimethylsilylation derivatization, and gas chromatography mass spectrometry was developed, partly validated, and applied to 18 surface sediments and a sediment core dated using the 210Pb method. The results showed the presence of the pharmaceuticals estriol, gemfibrozil, metoprolol, ketoprofen, naproxen, 17α-ethinylestradiol and estrone concentrated around the regions Napoleon Gulf and Thurston Bay with accumulation rates decreasing towards the top of the sediment core. Nonetheless, a randomness in the distribution of these compounds behooves a systematic assessment investigating not only the provenance of these compounds but also further investigations to errors meaning that this study should be treated as a preliminary investigation.

This study focuses on a general method that has been developed for the identification of both polar- and nonpolar pesticidespolar pesticides in a known solution from EPA 8151 Herbicide acid mix by Merck including the ordered non-polar pesticide Prosulfocarb. The EPA-solution contains 16 analytes that has been completely identified when derivatized and spiked in acetone. The solution has also been spiked in blood samples, resulting in five calibration solutions in the range of 0,01 – 2,5 µg/g, followed by quantification. Identification of dinoseb and bentazone, spiked in blood, failed whereas the remaining 145 analytes were identified. The method uses headspace (HS) solid phase microextraction (SPME) in combination with gas chromatography (GC) with mass spectrometry (MS) in both scan and selected ion monitoring mode. A standard extraction- and derivatization procedure has been performed for the analysis of alcohols, phenols, and carboxylic acids with help from a protocol regarding the analysis of ethylene glycol in blood. Samples were introduced on the column with splitless injection where 1 µl were injected with an injector temperature of 250oC. Effective separations were achieved by using GS-GasPro PLOT-column (30 m x i.d. 320 µm x df 0 µm) in combination with a temperature programme that started at an initial temperature of 80oC (1 min) that increased by 10oC/min up to 280oC (1 min). The limit of detection (LOD) for the pesticides, spiked blood, were 0,01 – 2,5 µg/g where the lowest limit of 0,01 µg/g meant difficult identification whereas a greater identification was made at 0,05 µg/g. No identification was succeeded for the most polar substances in the forms of amines and amides in combination with carboxylic acid. Identification was however made for the less polar pesticides in the forms of alcohols, phenols, and carboxylic acids. The method must be further developed to identify the highly polar pesticides in different chemical classes. The current method can be used in occurring intoxications and in autopsy cases.

No description available.