In forensic toxicology, opioids are frequently associated with drug abuse or drug-related death cases. An optimal method for use in the identification and quantification of opioids in a complex blood matrix is of paramount importance. Along with the ability to identify and quantitate opioids, this method should be accurate, sensitive, and selective. The application of sample pre-treatment and solid-phase extraction are common to purify and concentrate the target analytes before analyzing with liquid chromatography-tandem mass spectrometry. The purpose of this study was to compare the performance of two standard operating procedures, adopted by the Massachusetts State Police Crime Laboratory Toxicology and the Biomedical Forensic Sciences– Toxicology Laboratory at Boston University School of Medicine, for detecting opioids in blood. A total of eight drugs were analyzed: 6-monoacetylmorphine, codeine, fentanyl, hydrocodone, morphine, norhydrocodone, oxycodone, and oxymorphone. Comparison was performed using the parameters studied as part of method validation, including calibration model, bias, precision, carryover, interferences, ionization suppression/enhancement, and recovery. The results indicated that the method from Massachusetts State Police provided a better performance with between-run precision, interferences from matrix and other commonly encountered drugs, matrix effect at high concentration (250 ng/mL) and matrix recovery. Meanwhile, the method from Biomedical Forensic Sciences showed less bias, within-run precision, and matrix effect at low concentrations. Carryover and internal standard interference were comparable in both standard operating procedures. The calibration models were adjusted by altering the selection of regression model for improved quantification method performance. The volume of solvents, sample matrix, as well as time, were taken into consideration in accessing the overall performance of identification and quantitation. Both procedures were comparable yet the one from Massachusetts State Police was more beneficial in identifying the target analytes with greater sensitivity and selectivity and the one from Biomedical Forensic Sciences was more economical and efficient.

L'objectif principal de cette thèse est de mettre en évidence l'effet de la méthode de préparation sur la dynamique de relaxation des polymères en films minces. Nous nous sommes focalisés en particulier sur l'importance de la maîtrise du taux d'évaporation du solvant lors de la préparation des films, en termes de contrôle de la magnitude des contraintes résiduelles et les conséquences engendrées sur les propriétés physiques. Dans un premier temps, nous exposons la problématique de déviation des propriétés de polymères en films minces et le lien avec la technique de préparation. Nous décrivons un moyen simple permettant de contrôler le taux d'évaporation du solvant en ajustant la concentration de la solution et la vitesse de rotation de la tournette. Les expériences de démouillage nous ont permis de suivre l'effet du taux d'évaporation du solvant sur les propriétés viscoélastiques des polymères près de la Tg. Les résultats obtenus sur des films minces de polystyrène mettent en évidence le rôle de la méthode de préparation sur la probabilité de rupture des films, les temps caractéristiques de relaxation, la dynamique de démouillage et la magnitude des contraintes résiduelles. Dans une seconde partie, nous présentons également une expérience basée sur la propagation des fissures dans les films minces vitreux. Une simple observation microscopique, ainsi que des imageries en AFM permettent de prouver le rôle du taux d'évaporation du solvant lors de la préparation. Enfin, nous explorons une nouvelle approche expérimentale portant sur la possibilité d'investigations en temps réel et lors de démouillage la relaxation moléculaire des chaînes dans les films minces par des mesures diélectriques directes. Nous avons réussi à prouver non seulement un rôle de la méthode de préparation des films mais également que la restauration du comportement du polymère en volume « bulk » est possible en fonction du temps quelques soient les paramètres du film. Nous confirmons ainsi que les propriétés anormales observées dans les films minces spin-coatés sont dictées par l'état métastable provoqué par la méthode de préparation / The aim of this work is to highlight the influence of the sample preparation on the relaxation dynamics of supported polymer thin films. We focus in particular on the importance of tuning the solvent evaporation rate during films preparation, in terms of controlling the magnitude of residual stresses, and the impact on the physical properties. Firstly, we expose the deviation of the polymer behavior in thin polymer films related to the preparation technique. Then, we describe how the solvent evaporation rate can be precisely tuned by varying the concentration of the solution and the rotation rate of the spin coater. Dewetting experiments allowed us to investigate the effect of the solvent evaporation rate on the viscoelastic behavior of the polymers near the Tg. The probability of films rupture, the characteristic time, dewetting dynamics and the magnitude of residual stresses are deduced to prove the impact of sample preparation. In the second part, we present cracks propagation experiments in glassy thin films. A simple microscopic observation, as well as AFM imaging are used to emphasize the role of the solvent evaporation rate during the preparation. Finally, we present a new experimental approach to investigate in real time and during dewetting the dynamics of polymers in thin films by direct dielectric measurements. We have successfully proved not only the impact of sample preparation but also the restoration of the bulk behavior of polymers is possible during the time whatever the film parameters. We therefore confirm that the anomalous properties observed in spin-coated films are governed by the metastable state induced by the sample preparation

Injection molded short fiber reinforced composites have generated commercial interest in the manufacturing of lightweight parts used in semi-structural applications. Predicting these materials’ fiber orientation with quantitative accuracy is crucial for technological advancement, but the task is difficult because of the effect of inter-particle interactions at high concentrations of fiber found in parts of commercial interest. A complete sample preparation procedure was developed to obtain optical micrographs with optimal definition of elliptical and non-elliptical footprint borders. Two novel aspects in this procedure were the use of tridimensional markers to identify specific locations for analysis and the use of controlled-etching to produce small shadows where fibers recede into the matrix. These images were used to measure fiber orientation with a customized image analysis tool. This tool contains several modifications that we introduced in the method of ellipses which allow us to determine tridimensional fiber orientation and to obtain measurements in regions with fast changes in orientation. The tool uses the location of the shadow to eliminate the ambiguity problem in orientation and characterizes non-elliptical footprints to obtain the orientation in small sampling areas. Cavitywise measurements in two thin center-gated disks showed the existence of an asymmetric profile of orientation at the gate and an orientation profile that washed out gradually at the entry region until disappearing at about 32 gap widths. This data was used to assess the prediction of cavitywise orientation using a delay model for fiber orientation with model parameters obtained from rheometrical experiments. Model predictions combining slip correction and experimentally determined orientation at the gate are in agreement with experimental data for the core layers near the end-of-fill region. Radialwise measurements of orientation at the shell, transition and core layer, and microtextural description of the advancing front are included in this dissertation. The analysis and assessment of the radial evolution of fiber orientation and advancing front based on comparing the experimental data with simulation results are under ongoing investigation. / Ph. D.

Single Cell Proteomics (SCP) is an emerging discipline that contributes to a deeper understanding of individual cells' essential components. In biological systems, individual cells exhibit remarkable diversity, showcasing distinct proteomic profiles and functions. Mass Spectrometry (MS)-based techniques have become essential tools for exploring the proteomes of single cells with remarkable precision. While traditional bulk proteomics methods have been invaluable in revealing the overall protein composition of biological samples, they fall short in capturing the subtle nuances and heterogeneity among individual cells in a population. This limitation emphasizes the need for more targeted and detailed analyses to uncover the protein makeup of single cells. The MS-Based Single-Cell Proteomics technology serves as a valuable solution, providing comprehensive insight at the cellular level by analyzing proteins for identity, abundance, post-translational modifications, and interactions. This dissertation focuses on advancing single-cell proteomics through method development to enhance sensitivity and throughput. It presents a detailed protocol for a label-free single-cell proteomics workflow that integrates the cost-effective HP D100 Single Cell Dispenser and a one-hour, one-step sample preparation method. In contrast to the standard data-dependent acquisition method, the novel wide window acquisition (WWA) intentionally co-isolates and co-fragments adjacent precursors along with the selected precursor, using large isolation windows. Optimized WWA significantly increased the number of MS2-identified proteins by ≈40% compared to standard data-dependent acquisition. In a 40-minute LC gradient at ≈15 nL/min, an average of 3000 proteins per single HeLa cell was identified. Employing this platform, we compared protein expression in individual HeLa cells where the crucial autophagy gene, atg9a, was knocked out, and contrasted it with their isogenic wild-type parental line. To enhance throughput and robustness while preserving superior sensitivity at ultra-low flow rates, we developed an improved multi-column nanoLC system. This system features accelerated offline gradient generation, multiple storage sample loops with selective elution profiles, and allows for analysis as fast as every 20 minutes at 40 nL/min with close to 100% MS utilization time. Moreover, it enables continuous operation for up to 6 months without the need for column replacement. When applied to single-cell Multiple Myeloma treated with lenalidomide, this workflow identified an average of around 1300 unique protein groups.

No description available.

<p>Disease-specific compounds (biomarkers) are analyzed in clinical laboratories to assist with diagnosing diseases. This thesis describes development and validation of liquid chromatography tandem mass spectrometry (LC-MS/MS) based tests for diagnosing a diverse group of endocrine and metabolic diseases. The analytical methods used on-line and off-line sample extraction and analytical derivatization as means of enhancing the analytical sensitivity, specificity and clinical utility. All developed methods were extensively validated and reference intervals for the biomarker concentrations were established in blood samples of healthy adults and children. Advantages of the LC-MS/MS as an analytical technique include possibility of simultaneous measurement of multiple analytes and ability of confirming their identity. In this thesis we proposed and evaluated approaches for the assessment of the specificity of analysis in the methods that use tandem mass spectrometry detection. To enhance throughput of the LC-MS/MS tests for the biomarkers that have endogenous or exogenous isomers an approach was developed for quantitation of isomers from unresolved chromatographic peaks. Using methods developed in this thesis we performed a study of the steroidogenesis in ovarian follicles of healthy women and women with polycystic ovary syndrome (PCOS). Obtained data on the steroid concentrations and associations between the steroid metabolites in the pathway would be helpful for better understanding of the ovarian pathophysiology. Potential biomarkers of PCOS were identified in the thesis; further studies will be necessary to confirm their clinical utility.</p>

The polymer-like organic material in the Orgueil (Cl) chondrite was analyzed by high vacuum pyrolysis/gas chromatography/mass spectrometry, a new technique specially developed for this type of polymer analysis. Orgueil powder, previously solvent-extracted to remove all soluble organic compounds, whether indigenous or contaminant, was pyrolyzed in temperature steps at 150°, 300°, 450°, and 600°C. Gas chromatographic/mass spectrometric analysis of the vacuum pyrolyzates revealed a series of alkenes to C₈, an extensive series of alkylbenzene isomers, thiophene, alkylthiophenes, and benzothiophene, and the nitrogen compounds acetonitrile, acrylonitrile, and benzonitrile. Comparison of the Orgueil polymer fragments with those reported in polymer analyses of the Murchison and Allende meteorites suggests that qualitatively and quantitatively the Orgueil and Allende polymeric materials are very similar. The presence of acetonitrile, acrylonitrile, and benzonitrile, typical breakdown products of amino acids, in Orgueil pyrolyzates suggests the presence of amino acids in an insoluble form in the meteorite polymer matrix. Changes in the distribution of polymer fragments occurred during high vacuum pyrolyses as the temperature was increased stepwise from 150° to 600°C. These changes imply a progressive alteration in the character of the polymeric material toward a condensed aromatic structure through the preferential loss of aliphatic and heteroaromatic portions at lower temperatures. A possible inference is that the polymeric material in Orgueil is a complex mixture of polymerized materials having different thermal stability. Comparison of vacuum pyrolyzates of the Orgueil polymeric material with pyrolyzates of terrestrial kerogen indicates similarities in composition and structure. These similarities suggest the possibility that meteorite polymers may have formed near the surfaces of meteorite parent bodies by low temperature processes similar to those by which terrestrial kerogen is formed.

Contaminantes orgânicos há tempos são normalmente encontrados em baixíssimas concentrações no meio ambiente. Compostos orgânicos monoaromáticos, derivados dos subprodutos do petróleo, frequentemente são encontrados próximos a postos de combustíveis, onde há constantes vazamentos nos tanques de armazenamento. Poluentes emergentes, como exemplo os fármacos, também são temas constantes de pesquisas, devido à baixa taxa de degradação dos mesmos, mesmo após submetidos a sistemas de tratamento convencionais. Assim, a busca por técnicas de remediação, que visem a completa remoção de poluentes em baixas concentrações e metodologias analíticas que permitam o isolamento e detecção de tais compostos se torna essencial. Esse trabalho tem como uma de suas finalidades otimizar e aplicar um sistema de remoção de compostos orgânicos monoaromáticos presentes em solo, utilizando CO2 no estado supercrítico. A SPME-GC-MS foi otimizada e validada para a etapa de extração, com separação e quantificação por GC-MS dos contaminantes remanescentes após o solo ser submetido ao processo de remediação. Outro etapa do projeto, está relacionada com a determinação de sulfonamidas em lodo de esgoto. Para isso, um método rápido, fácil, barato e robusto de extração foi otimizado e validado, obtendo bons limites de quantificação (10-25 ng g-1) e detecção (4-9 ng g-1), com precisão e exatidão satisfatórios, de acordo com os valores estabelecidos pela ANVISA. Ainda com o propósito de determinar sulfonamidas em lodo de esgoto, um método LC online foi desenvolvido. A finalidade é promover o clean-up da matriz e extração dos analitos em um sistema de extração em fase sólida capilar online. Para isso, fez uso de duas colunas capilares (530 µm x 6,0 cm) dispostas em série, inseridas em um sistema cromatográfico capilar acoplado a detecção por espectrometria de massas. A primeira coluna teve como finalidade a retenção de interferentes presentes na amostra utilizando para isso, uma fase polimérica Strata-X. Já a segunda coluna, extraiu as sulfonamidas de interesse utilizando, para isso, uma fase polimérica aniônica Oasis Max. O extrato inserido no sistema de extração em fase sólida onlineera composto por 100 % solvente orgânico (acetonitrila); utilizando diferentes porcentagens de acetonitrila na etapa de carregamento, foi possível realizar clean-up adequado da amostra. A eluição dos analitos foi feita utilizando-se fase móvel acidificada. Com esse sistema, melhorou-se em até 30 % a extração de alguns compostos. Todos os métodos de preparo de amostra utilizados no trabalhos seguem a tendência atual sugerida pelo conceito do green chemistry. / The determination of organic contaminants in environmental matrices has long been a challenge for the scientific community, due to the very low concentrations they are present. Monoaromatic organiccompounds derived from petroleum sub-products are still a concern, because they are often found in environmental sites near gas stations, where there are constant leaks in storage tanks. Emerging pollutants,which include personal care products and pharmaceuticals, are also constant subject of research due to their low degradation rate, even after conventional treatment systems. Moreover, the research forremediation techniques aimed at complete removal of pollutants at low concentrations, as weel as new analytical methodologies for isolation and detection of such compounds are, both, essential. This workaims to optimize and apply a system to remove monoaromatic organic compounds present in soil, using CO2 in the supercritical state. As an analytical tool, SPME was optimized and validated with separation and quantification by GC-MS of selected contaminants remaining after the remediation process. Another research work developed is related to the determination of sulfonamides in sewage sludge.Firstly a methodology based on QuEChERS extraction method was optimized and validated, obtaining good results as limits of quantification, LOQ (10-25 ng g-1) and detection, LOD (4-9 ng g-1) withsatisfactory accuracy and precision, according to the values established by ANVISA. Still working to determine sulfonamides in sewage sludge, a new online sample preparation method was developed. Thepurpose of this method is to promote the matrix clean-up and concentration analytes in a capillary SPE system. It was used two capillary columns (500 µm x 6.0 cm) arranged in series in a capillarychromatographic system coupled to mass spectrometry. The first column was intended to retain interferences present in the sample, using a Strata-X polymer phase. The second column extracted sulfonamidesof interest, making use of an anionic polymer phase (Oasis MAX). The extract inserted in the online SPE system was composed of 100% organic solvent (acetonitrile); using different percentages of ACNwith appropriately pH at the loading stage, it was possible to achieve a satisfatory sample clean-up. The elution of the analytes was performed using an acidified mobile phase. This system improved by up to30% extraction yield of some compounds. All sample preparation methods used in the works follow the current trend suggested by the green chemistry concept.

O presente estudo analisou os efeitos da radiação gama na sobrevivência de fungos em plantas medicinais embaladas, adquiridas do comércio atacadista e varejista, em diferentes períodos (0 e 30 dias) após o tratamento por irradiação. Cinco tipos de plantas medicinais [Peumus boldus, Cameilia sinensis, Maytenus ilicifolia, Pauilinia cupana and Cássia angustifolia), foram coletadas de diferentes municípios do Estado de São Paulo e submetidas ao tratamento por irradiação, utilizando-se uma fonte 60Co (tipo Gammacell 220), com doses de 5,0 kGy e 10 kGy e taxa de dose de 3,0 kGy/h. Amostras não irradiadas (grupo controle) foram usadas na contagem de fungos e diluições seriadas de 10-1 a 10-6 das amostras foram semeadas em duplicata e plaqueadas usando o método de cultura em superfície, em ágar Dicloran Glicerol 18% (DG18) e contadas após cinco dias a 25°C. O grupo controle revelou a presença dos gêneros Aspergilius e Penicillium, os quais são conhecidos como fungos toxigênicos e poucas amostras do grupo controle estavam dentro dos limites seguros, estabelecidos pela Organização Mundial de Saúde (OMS, 1998) para plantas medicinais. Em resposta a resistência do tratamento por ionização, na dose de 5 kGy, foi obsen/ado que os gêneros Aspergilius, Piioma e Syncephalastrum foram radiorresistentes, após o processo (dia 0 e 30° dia). O tratamento por radiação gama foi efetivo na descontaminação de todas as amostras de plantas medicinais, após 30 dias, com a dose de 10 kGy e mantidas em condições de vedação. Não foram detectadas aflatoxinas nas amostras do grupo controle, ainda que estas amostras estivessem intensamente contaminadas com Aspergilius flavus. / This present study evaluated the effect of gamma radiation on the fungal survival in packed medicinal plants, purchased from wholesale and retail market, in different period (0 and 30 days) after the treatment. Five kind of medicinal plants (Peurnus boldus, Camellia sinensis, Maytenus ilicifolia, Pauilinia cupana and Cassia angustifolia), were collected from different cities of São Paulo State, and submitted to irradiation treatment using a 60Co source (type Gammacell 220) with doses of 5,0 kGy and 10 kGy and at dose rate of 3.0 kGy/h. Non-irradiated samples (control group) were used for fungal counts and serial dilutions from 10-1 to 10-6 of the samples were seeded in duplicates and plated using the surface culture method in Dichloran 18% Glycerol Agar (DG 18) and were counted after five days at 25°C. The control group revealed the presence of genera Aspergillus and Penicillium, which are known as toxigenic fungi and a few samples of control group were within the safety limits of World Health Organization (WHO, 1998) to medicinal plants. In response to resistance of ionizing treatment, in the dose of 5 kGy, it was obsen/ed that the genera Aspergillus, Phoma and Syncephalastrum were radio-resistant after the process (day 0 and 30° day). The treatment by gamma radiation was effective in decontamination of all irradiated samples of medicinal plants, after 30 days, with the dose of 10 kGy and kept of veiled conditions. It was not detected aflatoxins in samples of control group, even though these samples were heavily contaminated with Aspergillus flavus.

Propõe-se, pela primeira vez, a associação do tratamento fotocatalítico de amostra, direcionado à degradação e/ou eliminação de interferentes orgânicos, com a separação analito/matriz via geração de hidretos voláteis seguida de determinação por espectrometria de absorção atômica (HG-AAS). O fotocatalisador TiO2 (P-25) foi utilizado sempre em suspensão na amostra, e como fonte de radiação UV empregou-se lâmpada de mercúrio. Duas geometrias de reator fotocatalítico foram examinadas: irradiação estacionária de amostras contidas em cubetas de quartzo (3,5 mL); e irradiação em reator tubular constituído de bobina de Teflon montada em torno da lâmpada. Para avaliar a eficiência do tratamento sob diferentes condições, utilizou-se inicialmente o sistema-modelo Cd(II)-EDTA, com detecção do Cd(II) não quelado por voltametria de pulso diferencial em eletrodo de gota pendente de mercúrio, sabidamente não influenciada pelo TiO2 em suspensão. Nos estudos com HG-AAS, focalizou-se a aplicação do tratamento fotocatalítico à decomposição da arsenobetaína (Asb), um composto modelo interessante por ser refratário aos tratamentos convencionais (micro-ondas + digestão ácida). As condições selecionadas para a etapa de HG-AAS foram: concentração de HCl, 3 mol.L-1, concentração de NaBH4, 1% m/v em NaOH 0,1 mol.L-1, volume de amostra, 0,10 mL e temperatura do atomizador de tubo de quartzo, 980 °C. Nessas condições, as curvas de calibração obtidas por HG-AAS para As(III) e As(V) na faixa de 0,020 a 0,100 mg.L-1 (20 a 100 ppb) apresentaram linearidade e sensibilidade próximas, indicando eficiência de formação similares, característica esta favorável à quantificação total de arsênio em aplicações futuras a amostra reais. Estudos preliminares mostraram que a taxa de recuperação do arsênio é maior no tratamento fotocatalítico realizado em meio alcalino, (pH=12) em razão da menor tendência à adsorção de arsênio em TiO2 nesse meio frente ao neutro e ácido. Parâmetros como o tempo de irradiação, concentração do fotocatalisador e de Asb, além da influência de O2 molecular como scavenger de elétrons também foram investigados. Nas condições selecionadas, partindo-se de uma solução de Asb 0,075 mg.L-1 em arsênio, alcançou-se recuperação aproximada de 80% mediante irradiação por 15 minutos e de 100% após 45 minutos. A decomposição da Asb pelo método fotocatalítico proposto foi confirmada por espectrometria de massa com ionização por electrospray e estudos adicionais poderão revelar se as espécies de arsênio envolvidas na geração da arsina encontram-se completamente mineralizadas. / The combination of photocatalytic sample preparation, aiming degradation of organic interferences, with analyte/matrix separation by generation of volatile hydrides followed by atomic absorption spectrometric determination (HG-AAS) is proposed for the first time in this work, mainly devoted to the investigation of total arsenic analysis. A mercury lamp served as source of UV-radiation and the photocatalyst, TiO2 (P25), was used as a suspension in the sample solution. Two geometries of photocatalytic reactor were examined: stationary irradiation of a set of samples contained in 3.5 mL quartz cuvettes (preferred one for the present application) and stopped-flow irradiation of a sample in a tubular reactor consisting of a Teflon tube coiled around the lamp. Evaluation of the digestion efficiency of the reactors under different conditions was made with help of the model system Cd(II)-EDTA, with detection of the unchelated Cd(II) by differential pulse voltammetry with the hanging mercury drop electrode, known to work in the presence of TiO2 suspension. The subsequent studies in combination with HG-AAS focused on the photocatalytic degradation of arsenobetaine (Asb), chosen as a model because it is particularly resistant to conventional treatments (microwave + acid digestion). The following conditions were established for the HG-AAS step: HCl concentration (3 mol.L-1) and NaBH4 concentration (1% m/v in NaOH 0,1 mol.L-1), sample volume (0.10 mL) and atomization temperature (980 °C). Calibration curves for As (III) and As (V) by HG-AAS in the range of 0,020 to 0,100 mg.L-1 (20 a 100 ppb) conveyed in slope and linearity, indicating the same efficiency of arsine formation from both species, favoring the total quantification of arsenic in the sample. Alkaline medium (pH=12) was preferred for the photocatalytic digestion because recoveries of arsenic were better than in neutral or acidic medium, possibly due to lower losses by adsorption of arsenic species on TiO2. The effects of parameters such as irradiation time, concentration of the photocatalyst and of arsenobetaine, as well as the influence of molecular O2 as an electron scavenger were investigated. Under selected conditions and for a starting solution of 0,075 mg.L-1 Asb an arsenic recovery of 80% approximately was obtained after 15 minutes of irradiation while full recovery required 45 minutes. The decomposition of the Asb molecule after irradiation was confirmed by electrospray mass spectrometry and a further study may reveal if the arsenic species involved in the arsine generation are fully mineralized ones.