No description available.

Algae “super blooms” are a commonly encountered environmental issue in fresh and brackish water that occurs due to the buildup of cyanobacteria. Many of the commonly encountered cyanobacteria such as Mycrocystis aeruginosa (M. aeruginosa) produce potent cyanotoxins (microcystins) that pose serious health threats and even death to local wild life and humans. Microcystin contaminated fresh-water that empties into the ocean has been shown to lethally affect marine life in the area of contamination. Human consumption of tainted sea life and other routes of mycrocystin exposure can lead to serious liver damage and even death. Thus, a method was developed for forensic postmortem analysis of microcystins RR, LR and YR by Two-Dimensional (2D) Liquid Chromatography (LC) - tandem Mass Spectrometry (MS/MS). A final 2D LC-MS/MS method was selected from 6x6 automated method development experiments. Each microcystin were subjected to a total of 36 methods, which were completed over an 18hr period. The extraction process was performed using a reverse-phase sorbent (Oasis HLB, Waters Corporation, Milford, MA) with a 3cc solid phase extraction (SPE) barrel using sequential elution. From acetonitrile (ACN) and methanol (MeOH) stock solutions, 10 μL of the internal standard (IS) Nodularin was added to the final extract. The concept of sequential micro extraction was designed to capture the retention behaviour of the target analyte in response to various extraction parameters (sorbent strength, elution polarity, and solubility). Therefore, optimized conditions were selected to excise the region of interest during extraction. The elution solvents chosen for the microcystins were acetonitrile, methanol and acetone with 10 % sequential increments. Since microcystins exhibit a zwitterionic structure, three sets of elution solutions were created to evaluate their elution profile (pH 3, pH 7, pH 10). When the elution profile for low pH and high pH are compared, microcystin RR was eluted over the 40% to 70% methanol fractions under low pH conditions with a slight shift towards higher organic % (50%-70% fractions) under high pH. This elution behaviour suggests that the basic moieties of the structure demonstrate a stronger retention for the stationary phase. Microcystin LR and YR however, eluted at a higher organic solvent percentage under low pH conditions and at a lower organic solvent percentage under high pH conditions, indicates that the acidic moieties of the structures have stronger retention. The urine sample gave recovery values for all three microcystins in the 80-90% range, as to be expected with type of complexity associated with biological samples. The sequential extraction protocol produced an extraction method that delivered a clean extract after a 30 min workflow using a single and optimized 2D LC-MS/MS method. The total analytical run time was set at 10 minutes.

Le cancer colorectal (CCR) reste encore aujourd'hui une cause majeure de mortalité dans le monde, avec une incidence annuelle d'environ 1 million de cas et une mortalité d'environ 700 000 personnes. La stratégie de dépistage du CCR se base sur la détection de sang occulte dans les selles par le test iFOBT. Malgré une spécificité acceptable, la sensibilité de détection reste toujours insuffisante dans une population asymptomatique (27% de sensibilité pour un adénome, 65% pour un CCR). Aujourd'hui, il existe un réel besoin de trouver de nouveaux biomarqueurs spécifiques du CCR ainsi que de développer des tests de diagnostic faciles à mettre en oeuvre dans les laboratoires de biologie médicale. Les progrès récents de l'analyse protéomique à haut débit permettent aujourd'hui d'identifier et de quantifier un nombre toujours plus grand de protéines. C'est pourquoi, nous avons développé des stratégies en protéomique quantitative pour la recherche et la validation de biomarqueurs du cancer colorectal. L'approche iTRAQ et Label-free ont permis l'identification d'environ 800 protéines et la quantification de 214 protéines. Nous avons entrepris avec succès, la validation de 4 protéines par des tests ELISA et de nouvelles technologie en protéomique quantitative ciblée (LC-SRM). Au Final, nous proposons un test multiplex de 3 protéines qui permettra d'affirmer ou d'infirmer les résultats obtenus par le test iFOBT. L'ajout du test pourrait réduire de manière significative le nombre de coloscopies inutiles, potentiellement dangereuses pour les patients et onéreuses pour les autorités de santé. / Colorectal cancer (CRC) remains a major cause of cancer mortality throughout the world with an annual incidence of approximately 1 million cases and an annual mortality around 700 000. CRC outcome is highly dependent upon the stage of detection. The 5-year survival rate of patients with metastases is less than 20% while patients with localized adenomatous polyps have an excellent outcome with 90% survival rate. Current screening methods of CRC include the iFOBT and colonoscopy. The iFOBT has an acceptable 95% specificity but always an unsatisfactory sensitivity of detection in the asymptomatic population (27% sensitivity for adenoma, 65% for carcinoma). That is why, there is a real need to find new biomarkers and develop new diagnostic test, specific and easy to implement in clinical research. Recent advances in the analysis of biological fluids by high throughput mass spectrometry allow us now to identify and quantify a large number of proteins. Due to these improvements of proteomic strategies, it seems to be a promising way to find new potential proteins markers in the CRC disease. The quantitative iTRAQ & Label-free approaches allowed the identification of 800 proteins and the quantification of 200 proteins. Therefore, we have successfully validated four proteins by targeted proteomics using mass spectrometry (LC-SRM) and ELISA assays. Finally, we suggest a multiplex test of 3 proteins which confirm or contradict the FOBT outcome. The addition of the test could significantly reduce the number of unnecessary colonoscopies which are potentially dangerous for patients and expensive for public health authorities.

none

The issue with maintaining stability of compounds during storage and transportation has been universal across many fields such as clinical, pharmaceutical, food industry and especially in forensic laboratories where case backlogs hinder immediate analysis of samples upon collection. This study compares various storage conditions and analyzes changes in compound stability over 28 days. The various storage conditions compared in this study are temperature (+4 °C and -20 °C); water sample and urine sample and solid state (loading onto Solid Phase Extraction cartridges) and storing in its liquid state (present in the urine or water sample). 51 compounds were analyzed in this study belonging to pharmaceutical and hormone classes. Two Dimensional Liquid Chromatography was used for separation allowing the analysis of varied compounds in terms of chemistry. Mass Spectrometry was used for detection of these compounds. This study conclusively helped determine that there is no significant difference in stability of 42 out of 51 of these compounds on comparing them in solid versus liquid state over a period of 28 days. This helps determine that Solid Phase Extraction (SPE) cartridges can be used as an alternative for storage and transportation of these compounds. 46 compounds showed no significant difference in +4 and -20 °C temperature storage conditions as well. Despite not including a wash step during sample preparation between loading and elution, 11 out of 51 compounds did not show suppression or enhancement due to matrix effect. This study not only highlights the importance of sample preparation prior to analysis but also shows how SPE technique could help maintain stability of compounds during storage. In future studies, stability changes using SPE for long term storage could provide beneficial results to conclude if SPE techniques can provide an advantage over liquid state for a period longer than a month.

Made available in DSpace on 2016-01-26T18:56:03Z (GMT). No. of bitstreams: 1 Deigilam Cestari Esteves.pdf: 2776358 bytes, checksum: 7e1a46b5b264cbf8e0c0d96e68b501e2 (MD5) Previous issue date: 2014-12-18 / The environmental and hospital infections, caused by bacteria which are resistant to a wide variety of antibiotics, have shown increasing records in the last years, manifesting themselves in high mortality and lethality. Recent researches report that the bacteria presents a survival profile on dry surfaces in order to maintain their virulence when exposed to biological fluids such as urine, saliva and blood. The objective of this study was to document - through laboratorial analysis the survival capacity of the main bacteria of medical interest on abiotic surfaces. The adopted procedures were completely conducted in the microbiology laboratory at Unoeste, in Presidente Prudente SP. Standard ATCC strains of Staphylococcus aureus (ATCC25923), Enterococcus faecalis (ATCC29212), Escherichia coli (ATCC35218), Klebsiella pneumoniae (ATCC700603) and Pseudomonas aeruginosa (ATCC27583) were used, to which biological fluids and water were added. The surfaces were tile, synthetic fabric, mattress and cotton fabric. The surfaces were contaminated with suspension composed by the ATCC strains, biological fluids and water in addition to the control containing only the ATCC strains, which were stored in petri dishes, kept in room temperature. Every seven days the surfaces were dipped in trypticase soy broth and kept in the oven for 24h at 37ºC. They were spread from the contaminated brothin Mueller Hinton agar and kept for 24h in the oven at 37ºC. The viability analysis was done through the colony-forming unity (CFU) counting. For the statistical analysis, the Software R was used, through the Friedman and Kruskal Wallis tests for multiple comparisons, analyzing within each factor (solution or surface) which differ between one another (Friedman s test) and also which treatments interactions are different (Kruskal-Wallis). Through this analysis it was possible to observe that in the current work that Staphylococcus aureus kept its viability for a longer period than other microorganisms under all the tested conditions, presenting significant differences between the fluids and surfaces, with a particular colony growth in blood and cotton. The Klebsiella pneumoniae showed differences between fluids, with saliva containing the highest colony number. With regards to the other tested bacteria, there was no isolated significance. For the multiple comparisons analysis, only the Pseudomonas aeruginosa presented no significant difference between any pair of treatments, whereas the other bacteria presented significant differences between the correlations. The need to analyze the environmental impact of these microorganisms persistence in environments, which are vulnerable to human beings, guides the creation of measures in order to control the spread of pathogenic microorganisms. / As infecções ambientais e hospitalares causadas por bactérias resistentes a um amplo espectro de antibióticos têm índices crescentes nos últimos anos, manifestando-se com alta morbidade e letalidade. Pesquisas recentes evidenciam que as bactérias demonstram um perfil de sobrevivência, em superfícies secas de modo a manter sua virulência quando expostas a fluidos biológicos como urina, saliva e sangue. O objetivo desse estudo foi documentar através de análises laboratoriais a capacidade de sobrevivência das principais bactérias de interesse médico em superfícies abióticas. Os procedimentos foram totalmente realizados no laboratório de microbiologia da Unoeste em Presidente Prudente - SP., utilizando cepas padrão ATCC de Staphylococcus aureus(ATCC25923), Enterococcus faecalis (ATCC29212), Escherichia coli(ATCC35218), Klebsiella pneumoniae(ATCC700603) e Pseudomonas aeruginosa(ATCC27583), ao qual foram adicionados fluídos biológicos e água. As superfícies utilizadas foram piso, tecido sintético, colchão e tecido algodão. As superfícies foram contaminadas com suspensão composta pelas cepas ATCC, fluídos biológicos e água, além do controle contendo somente as cepas ATCC, armazenadas em placas de petri e mantidas em temperatura ambiente. A cada sete dias as superfíciesarmazenadas foram mergulhadas em caldo Tripscaseína de Soja (TSB), colocadas na estufa por 24h a 37ºC. Foram semeados do caldo contaminado em ágar Mueller Hinton e mantidos por 24h em aquecimento a 37º em Estufa.A análise da viabilidadefoi realizada através da contagem de unidade formadora de colônias (U.F.C.). Para a análise estatística utilizou o software R, realizando os testes de Friedman e de Kruskal Wallis para comparações multiplas, analisando dentro de cada fator (solução ou superfície) quais diferem entre si (Teste de Friedman) e, também, quais interações de tratamentos são diferentes (Kruskal-Wallis). Através dessas análises foi possível observar no presente trabalho que Staphylococcus aureus manteve a viabilidade por tempo maior que os outros microrganismos em todas as condições testadas, apresentando diferença significativa entre os fluídos e as superfícies, tendo sangue e tecido algodão crescimento de maior número de colônias. A Klebsiella pneumoniae apresentou diferença entre os fluídos, sendo asaliva com maior número de colônias. Para as outras bactérias testadas não houve significância isoladamente. Para as análises de comparações múltiplas somente para a Pseudomonas aeruginosanão houve diferença significativa entre algum par de tratamentos, as outras bactérias apresentaram diferenças significativas entre as correlações. A necessidade de analisar o impacto ambiental da persistência desses microrganismos em ambientes vulneráveis ao ser humano, norteia o delineamento de medidas para o controle na disseminação de microrganismos patogênicos.

Made available in DSpace on 2016-07-18T17:46:20Z (GMT). No. of bitstreams: 1 Deigilam Cestari Esteves.pdf: 2776358 bytes, checksum: 7e1a46b5b264cbf8e0c0d96e68b501e2 (MD5) Previous issue date: 2014-12-18 / The environmental and hospital infections, caused by bacteria which are resistant to a wide variety of antibiotics, have shown increasing records in the last years, manifesting themselves in high mortality and lethality. Recent researches report that the bacteria presents a survival profile on dry surfaces in order to maintain their virulence when exposed to biological fluids such as urine, saliva and blood. The objective of this study was to document - through laboratorial analysis the survival capacity of the main bacteria of medical interest on abiotic surfaces. The adopted procedures were completely conducted in the microbiology laboratory at Unoeste, in Presidente Prudente SP. Standard ATCC strains of Staphylococcus aureus (ATCC25923), Enterococcus faecalis (ATCC29212), Escherichia coli (ATCC35218), Klebsiella pneumoniae (ATCC700603) and Pseudomonas aeruginosa (ATCC27583) were used, to which biological fluids and water were added. The surfaces were tile, synthetic fabric, mattress and cotton fabric. The surfaces were contaminated with suspension composed by the ATCC strains, biological fluids and water in addition to the control containing only the ATCC strains, which were stored in petri dishes, kept in room temperature. Every seven days the surfaces were dipped in trypticase soy broth and kept in the oven for 24h at 37ºC. They were spread from the contaminated brothin Mueller Hinton agar and kept for 24h in the oven at 37ºC. The viability analysis was done through the colony-forming unity (CFU) counting. For the statistical analysis, the Software R was used, through the Friedman and Kruskal Wallis tests for multiple comparisons, analyzing within each factor (solution or surface) which differ between one another (Friedman s test) and also which treatments interactions are different (Kruskal-Wallis). Through this analysis it was possible to observe that in the current work that Staphylococcus aureus kept its viability for a longer period than other microorganisms under all the tested conditions, presenting significant differences between the fluids and surfaces, with a particular colony growth in blood and cotton. The Klebsiella pneumoniae showed differences between fluids, with saliva containing the highest colony number. With regards to the other tested bacteria, there was no isolated significance. For the multiple comparisons analysis, only the Pseudomonas aeruginosa presented no significant difference between any pair of treatments, whereas the other bacteria presented significant differences between the correlations. The need to analyze the environmental impact of these microorganisms persistence in environments, which are vulnerable to human beings, guides the creation of measures in order to control the spread of pathogenic microorganisms. / As infecções ambientais e hospitalares causadas por bactérias resistentes a um amplo espectro de antibióticos têm índices crescentes nos últimos anos, manifestando-se com alta morbidade e letalidade. Pesquisas recentes evidenciam que as bactérias demonstram um perfil de sobrevivência, em superfícies secas de modo a manter sua virulência quando expostas a fluidos biológicos como urina, saliva e sangue. O objetivo desse estudo foi documentar através de análises laboratoriais a capacidade de sobrevivência das principais bactérias de interesse médico em superfícies abióticas. Os procedimentos foram totalmente realizados no laboratório de microbiologia da Unoeste em Presidente Prudente - SP., utilizando cepas padrão ATCC de Staphylococcus aureus(ATCC25923), Enterococcus faecalis (ATCC29212), Escherichia coli(ATCC35218), Klebsiella pneumoniae(ATCC700603) e Pseudomonas aeruginosa(ATCC27583), ao qual foram adicionados fluídos biológicos e água. As superfícies utilizadas foram piso, tecido sintético, colchão e tecido algodão. As superfícies foram contaminadas com suspensão composta pelas cepas ATCC, fluídos biológicos e água, além do controle contendo somente as cepas ATCC, armazenadas em placas de petri e mantidas em temperatura ambiente. A cada sete dias as superfíciesarmazenadas foram mergulhadas em caldo Tripscaseína de Soja (TSB), colocadas na estufa por 24h a 37ºC. Foram semeados do caldo contaminado em ágar Mueller Hinton e mantidos por 24h em aquecimento a 37º em Estufa.A análise da viabilidadefoi realizada através da contagem de unidade formadora de colônias (U.F.C.). Para a análise estatística utilizou o software R, realizando os testes de Friedman e de Kruskal Wallis para comparações multiplas, analisando dentro de cada fator (solução ou superfície) quais diferem entre si (Teste de Friedman) e, também, quais interações de tratamentos são diferentes (Kruskal-Wallis). Através dessas análises foi possível observar no presente trabalho que Staphylococcus aureus manteve a viabilidade por tempo maior que os outros microrganismos em todas as condições testadas, apresentando diferença significativa entre os fluídos e as superfícies, tendo sangue e tecido algodão crescimento de maior número de colônias. A Klebsiella pneumoniae apresentou diferença entre os fluídos, sendo asaliva com maior número de colônias. Para as outras bactérias testadas não houve significância isoladamente. Para as análises de comparações múltiplas somente para a Pseudomonas aeruginosanão houve diferença significativa entre algum par de tratamentos, as outras bactérias apresentaram diferenças significativas entre as correlações. A necessidade de analisar o impacto ambiental da persistência desses microrganismos em ambientes vulneráveis ao ser humano, norteia o delineamento de medidas para o controle na disseminação de microrganismos patogênicos.

Un biomarqueur est une molécule (ou un ensemble de molécules) présente dans l’organisme qui témoigne de l’apparition d’un processus pathologique. Il permet ainsi de dépister une maladie, d’en prédire sa gravité ou encore d’évaluer l’efficacité d’un traitement. Les liquides biologiques représentent des milieux de choix pour la recherche de biomarqueurs en pathologie humaine car leur collection est habituelle dans la prise en charge des patients et moins invasive comparée aux biopsies d’organes ou de tissus. Dans cette thèse, nous nous sommes intéressés plus particulièrement aux exosomes présents dans ces liquides biologiques. Les exosomes sont des nanovésicules dont le diamètre est compris entre 30 et 100 nanomètres. Ils sont sécrétés par tous les types cellulaires et contiennent des protéines cytoplasmiques et membranaires spécifiques de leur cellule d’origine. L’intérêt majeur des exosomes isolés à partir des liquides biologiques, est qu’ils constituent une source de biomarqueurs. Ils peuvent donc être assimilés à une « biopsie liquide ». L’analyse des exosomes pourrait compléter utilement des examens classiques de dépistage, de diagnostic et de suivi d’une pathologie. Dans le cadre de projet de cette thèse, nous avons appliqué des techniques de protéomique à haut débit pour l’analyse des exosomes. Nous nous sommes tout d’abord intéressés à l’analyse du profil protéique des exosomes urinaires dans le contexte de deux pathologies du tractus urinaire : la cystinurie et le cancer du rein. La cystinurie est une néphropathie lithiasique d’origine génétique pour laquelle il y a peu de marqueurs biologiques pouvant prédire son évolution vers l’insuffisance rénale terminale. Nous avons développé une méthode de préparation des exosomes urinaire permettant d’analyser de façon reproductible leurs profils protéiques. Nous avons appliqué cette méthode à huit patients cystinuriques et comparé les résultats aux profils obtenus chez dix sujets sains. Un panel de 38 protéines différentiellement exprimé dans les exosomes des patients a été identifié et en partie validé par Western blot. Concernant le cancer du rein à cellules claires pour lequel le diagnostic nécessite des prélèvements invasifs par biopsie, nous avons analysé les exosomes urinaires de huit patients avant et après néphrectomie. Nous avons ainsi pu mettre en évidence un panel de 25 protéines surexprimées dans les exosomes des patients. Enfin, le dernier volet de cette thèse a été consacré à l’analyse des exosomes du lavage broncho-alvéolaire provenant de patients MV, maladie d’origine génétique qui atteint principalement les poumons. L’analyse des exosomes de lavage broncho-alvéolaire pourrait permettre de donner un éclairage nouveau sur la physiopathologie de la maladie. Nous avons réalisé la comparaison des profils protéiques des exosomes de quatre patients MV, et six patients asthmatiques. L’ensemble des résultats obtenus au cours de cette thèse montre que l’analyse protéomique des exosomes issus de fluides biologiques peut aider la recherche de biomarqueurs diagnostics ou pronostics de maladies. / A biomarker is a molecule (or a cluster of molecule) which will reflect the occurrence of a pathological state, giving us the ability to detect a disease, to predict its severity or to assess drug efficiency. Biological fluids are the golden standards for biomarker research in human as they are routinely collected for patients’ follow-up and are less invasive than biopsies. During my PhD, I focused on exosomes that can be found in these biological fluids. Exosomes are nanovesicles with a diameter ranging between 30 and 100 nanometers. Exosomes are secreted by all cell types and harbor cytoplasmic and membranous proteins specific of their cells of origins. One of the major interest of exosomes enriched from biological fluids is that they represent a valuable source of biomarkers. They can be considered as a « liquid biopsy ». Their analysis could complete classical diagnosis and follow-up tools. In this project, we applied high resolution, high throughput proteomic techniques for exosomes analysis. We firstly focused on protein profiles in urinary exosomes in the context of two urinary tract diseases: cystinuria and kidney cancer. Cystinuria is an inherited autosomal recessive disease that is characterized by the formation of cystine stones in the kidneys. To date, there are no markers to predict the evolution toward end stage renal disease. We developed a method to prepare exosomes in order to reproducibly analyze their protein profiles. We applied this method to eight cystinuria patients and compared their profiles to those of ten healthy subjects. A panel of 38 differentially expressed proteins in patients were found and validated by western blots. We also applied this method to patients with clear cell renal cell carcinoma, for which invasive biopsies are necessary for clear diagnosis. We analyzed urinary exosomes form eight patients before and after nephrectomy. We were able to highlight 25 overexpressed proteins in patients’ exosomes. Eventually, the last part of my thesis was dedicated to the analysis of exosomes enriched from bronchoalveolar lavage fluid collected in cystic fibrosis patients, a disease that affects mostly the lungs. Bronchoalveolar lavage fluid exosomes analysis could give a new insight on the mechanisms of this disease. We compared protein profiles in exosomes from four cystic fibrosis patients and six asthmatic patients. The whole point of this work is to show that proteomic analysis of exosomes isolated from biological fluids could become a golden standard for the discovery of diagnosis or prognosis biomarkers.

O preparo da amostra é uma das etapas mais importantes em toda a análise química. O isolamento e a concentração dos componentes da amostra são cruciais e busca-se sempre que essas etapas sejam as mais simples e consumam o mínimo possível de tempo e reagentes. Nos últimos anos, um tipo de material tem se mostrado bastante útil para análises químicas a partir de fluidos biológicos, os polímeros molecularmente impressos (MIPs). Os MIPs são sintetizados por reações de polimerização, na presença de uma molécula molde (template). A molécula molde se liga aos monômeros funcionais do polímero durante a reação de polimerização e permanece ligada à superfície das cadeias poliméricas quando a reação se completa. Terminada a polimerização, realiza-se a completa lavagem das moléculas molde, assim, restam na superfície polimérica cavidades tridimensionais complementares à molécula empregada como molde. Essas cavidades permitem a ligação reversível e preferencial da molécula molde ou outras com estrutura química semelhante. A Cannabis sativa é a droga ilícita mais consumida em todo o mundo e nos últimos anos muita atenção tem se voltado a seus efeitos toxicológicos no corpo humano e a aplicações medicinais. Nesta dissertação, foi sintetizado um MIP com a molécula molde catequina para a extração e posterior análise por LC-MS/MS dos canabinóides Δ9-tetrahidrocanabinol (THC), 11-hidroxi-Δ9-tetrahidrocannabinol (THC-OH) e 11-nor-Δ9-tetrahidrocannabinol-9-ácido carboxílico (THC-COOH) em amostras de urina. O MIP produzido foi empacotado em microdispositivo e empregado no preparo das amostras de urina por microextração por sorvente empacotado (MEPS). O método desenvolvido apresentou boa linearidade (valores de r de 0,977 para o THC e 0,994 para THC-OH e THC-COOH). Os limites de detecção e de quantificação foram respectivamente de 5 ng mL-1 e 20 ng mL-1, para os compostos THC e THC-OH, na faixa linear de 25 a 250 ng mL-1. Para o composto THC-COOH os limites de detecção e quantificação alcançados foram de 1 ng mL-1 e 5 ng mL-1, respectivamente, na faixa linear de 5 a 170 ng mL-1. O método apresentou valores razoáveis de precisão entre 3,2% (THC-COOH) e 25,1% (THC) e de exatidão, que variou entre -18,4 e 17,4 (ambos para o THC). O MIP empregado no preparo da amostra mostrou-se mais seletivo e específico do que materiais normalmente empregados para a extração dos canabinoides das amostras de urina, além de a técnica de extração por MEPS apresentar baixo consumo de solventes e amostra para a extração dos analitos e posterior análise por LC-MS/MS. / The sample preparation is one of the most important steps in every chemical analysis. The isolation and concentration of the sample components are crucial and it is always sought that these steps are simple and consume the lowest amount of time and reagents. In the recent years, a type of material has proved to be very useful for chemical analyzes of biological fluids, the molecularly imprinted polymers (MIPs). MIPs are synthesized by polymerization reactions in the presence of a template molecule. The template molecule binds to the functional monomers of the polymer during the polymerization reaction and remains bonded to the surface of the polymeric chains after the reaction is complete. After the polymerization is finished, the complete washing of the template molecules is carried out, thus, three-dimensional cavities, complementary to the molecule used as a template, remain on the polymer surface. These cavities allow the reversible and preferential bonding of the template molecule or others with similar chemical structure. Cannabis sativa is the most commonly consumed illicit drug in the world and in recent years much attention has focused on its toxicological effects on human body and for medical applications. In this master dissertation, a MIP was synthesized with the catechin molecule as template, for extraction and subsequent analysis by LC-MS/MS of the cannabinoids Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. The MIP produced was packed in a microdevice and used in the preparation of the urine samples by microextraction by packed sorbent (MEPS). The developed method showed good linearity (r values of 0.977 for THC and 0.994 for THC-OH and THC-COOH). The detection and quantification limits were respectively 5 ng mL-1 and 20 ng mL-1 for THC and THC-OH in the linear range from 25 to 250 ng mL-1. For the compound THC-COOH the limits of detection and quantification achieved were 1 ng mL-1 and 5 ng mL-1, respectively, in the linear range from 5 to 170 ng mL-1. The method presented reasonable values of precision, between 3.2% (for THC-COOH) and 25.1% (for THC) and displayed accuracy ranging from -18.4 to 17.4 (both for THC). The MIP used in the sample preparation was more selective and specific than other materials usually employed for the extraction of the cannabinoids from the urine samples. The MEPS technique also showed low consumption of solvents and sample for sample preparation, extraction of analytes and subsequent analysis by LC-MS/MS.

O preparo da amostra é uma das etapas mais importantes em toda a análise química. O isolamento e a concentração dos componentes da amostra são cruciais e busca-se sempre que essas etapas sejam as mais simples e consumam o mínimo possível de tempo e reagentes. Nos últimos anos, um tipo de material tem se mostrado bastante útil para análises químicas a partir de fluidos biológicos, os polímeros molecularmente impressos (MIPs). Os MIPs são sintetizados por reações de polimerização, na presença de uma molécula molde (template). A molécula molde se liga aos monômeros funcionais do polímero durante a reação de polimerização e permanece ligada à superfície das cadeias poliméricas quando a reação se completa. Terminada a polimerização, realiza-se a completa lavagem das moléculas molde, assim, restam na superfície polimérica cavidades tridimensionais complementares à molécula empregada como molde. Essas cavidades permitem a ligação reversível e preferencial da molécula molde ou outras com estrutura química semelhante. A Cannabis sativa é a droga ilícita mais consumida em todo o mundo e nos últimos anos muita atenção tem se voltado a seus efeitos toxicológicos no corpo humano e a aplicações medicinais. Nesta dissertação, foi sintetizado um MIP com a molécula molde catequina para a extração e posterior análise por LC-MS/MS dos canabinóides Δ9-tetrahidrocanabinol (THC), 11-hidroxi-Δ9-tetrahidrocannabinol (THC-OH) e 11-nor-Δ9-tetrahidrocannabinol-9-ácido carboxílico (THC-COOH) em amostras de urina. O MIP produzido foi empacotado em microdispositivo e empregado no preparo das amostras de urina por microextração por sorvente empacotado (MEPS). O método desenvolvido apresentou boa linearidade (valores de r de 0,977 para o THC e 0,994 para THC-OH e THC-COOH). Os limites de detecção e de quantificação foram respectivamente de 5 ng mL-1 e 20 ng mL-1, para os compostos THC e THC-OH, na faixa linear de 25 a 250 ng mL-1. Para o composto THC-COOH os limites de detecção e quantificação alcançados foram de 1 ng mL-1 e 5 ng mL-1, respectivamente, na faixa linear de 5 a 170 ng mL-1. O método apresentou valores razoáveis de precisão entre 3,2% (THC-COOH) e 25,1% (THC) e de exatidão, que variou entre -18,4 e 17,4 (ambos para o THC). O MIP empregado no preparo da amostra mostrou-se mais seletivo e específico do que materiais normalmente empregados para a extração dos canabinoides das amostras de urina, além de a técnica de extração por MEPS apresentar baixo consumo de solventes e amostra para a extração dos analitos e posterior análise por LC-MS/MS. / The sample preparation is one of the most important steps in every chemical analysis. The isolation and concentration of the sample components are crucial and it is always sought that these steps are simple and consume the lowest amount of time and reagents. In the recent years, a type of material has proved to be very useful for chemical analyzes of biological fluids, the molecularly imprinted polymers (MIPs). MIPs are synthesized by polymerization reactions in the presence of a template molecule. The template molecule binds to the functional monomers of the polymer during the polymerization reaction and remains bonded to the surface of the polymeric chains after the reaction is complete. After the polymerization is finished, the complete washing of the template molecules is carried out, thus, three-dimensional cavities, complementary to the molecule used as a template, remain on the polymer surface. These cavities allow the reversible and preferential bonding of the template molecule or others with similar chemical structure. Cannabis sativa is the most commonly consumed illicit drug in the world and in recent years much attention has focused on its toxicological effects on human body and for medical applications. In this master dissertation, a MIP was synthesized with the catechin molecule as template, for extraction and subsequent analysis by LC-MS/MS of the cannabinoids Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. The MIP produced was packed in a microdevice and used in the preparation of the urine samples by microextraction by packed sorbent (MEPS). The developed method showed good linearity (r values of 0.977 for THC and 0.994 for THC-OH and THC-COOH). The detection and quantification limits were respectively 5 ng mL-1 and 20 ng mL-1 for THC and THC-OH in the linear range from 25 to 250 ng mL-1. For the compound THC-COOH the limits of detection and quantification achieved were 1 ng mL-1 and 5 ng mL-1, respectively, in the linear range from 5 to 170 ng mL-1. The method presented reasonable values of precision, between 3.2% (for THC-COOH) and 25.1% (for THC) and displayed accuracy ranging from -18.4 to 17.4 (both for THC). The MIP used in the sample preparation was more selective and specific than other materials usually employed for the extraction of the cannabinoids from the urine samples. The MEPS technique also showed low consumption of solvents and sample for sample preparation, extraction of analytes and subsequent analysis by LC-MS/MS.