There has been an emergence of novel psychoactive substances (NPS) in forensic casework globally. Although the reported prevalence of these compounds has been relatively low in comparison to traditional drugs of abuse, published case studies suggest that some NPS have significant pharmacological effects that may cause severe impairment and/or death. Because of these effects, it is important that toxicology laboratories have the capability of identifying these compounds to complete a comprehensive toxicological analysis for human performance and post-mortem investigations. Recently, mass spectrometry has gained favor over traditional screening assays such as immunoassays for the identification of NPS in biological specimens. This trend is mainly a result of the fact that mass spectrometry provides the required sensitivity and selectivity for a broader range of analytes. High resolution tandem mass spectrometry has been suggested for analysis of NPS, as this technique further increases selectivity by increasing mass accuracy and providing MS/MS spectral data. The main goal of the present study was to investigate the applicability of using high resolution mass spectrometry to screen for and confirm a large number of novel psychoactive substances. The present study consisted of three main tasks, which included 1) the creation of a large high resolution MS/MS spectral library and database, 2) the development of a solid phase extraction (SPE) method and acquisition methods, and 3) a collision induced dissociation (CID) study of regioisomeric NPS compounds. The MS/MS spectral library created contains spectral data for 252 NPS. In addition, 875 NPS entities were included in the compound database. The library and database can be used by toxicology laboratories to aid in the identification of NPS in casework using MS/MS spectral data and full scan MS data, respectively. The analytical method developed used SPE and high resolution mass spectrometry (HRMS). The HRMS method demonstrated limits of detection ranging from 0.5- 5 ng/mL for NPS from various structural drug classes. The CID experiments demonstrated that relative ion abundance alone could be used to differentiate some sets of regioisomers. The present work can aid toxicology laboratories in the identification of NPS and demonstrates the applicability of HRMS for their screening and confirmation.

LOPES NETO, José Joaquim. Bioacessibilidade e biodisponibilidade de compostos fenólicos de sementes de Triplaris Gardneriana Wedd (Polygonaceae). 2017. 95 f. Dissertação (Mestrado em Bioquímica)- Universidade Federal do Ceará, 2017. / Submitted by Weslayne Nunes de Sales (weslaynesales@ufc.br) on 2017-07-10T12:20:53Z No. of bitstreams: 1 2017_dis_jjlopesneto.pdf: 1657114 bytes, checksum: 3ec381c68cd046900e25b007ed35226a (MD5) / Approved for entry into archive by Weslayne Nunes de Sales (weslaynesales@ufc.br) on 2017-07-11T11:56:07Z (GMT) No. of bitstreams: 1 2017_dis_jjlopesneto.pdf: 1657114 bytes, checksum: 3ec381c68cd046900e25b007ed35226a (MD5) / Made available in DSpace on 2017-07-11T11:56:07Z (GMT). No. of bitstreams: 1 2017_dis_jjlopesneto.pdf: 1657114 bytes, checksum: 3ec381c68cd046900e25b007ed35226a (MD5) Previous issue date: 2017 / Triplaris gardneriana Wedd is a plant species from Brazilian semiarid region with seeds rich in phenolic compounds and high antioxidant capacity.The consumption of fruits and vegetables has been associated to low incidence of chronic diseases caused by oxidative stress. Among the food components capable of combating excess free radicals arethe phenolic compounds, with antioxidant capacity, which have been extensively studied. However, the total quantity of these substances present in foods does not necessarily reflect the bioactive amount absorbed and metabolized by the body. In this context, the present work aimed the development of an ethanolic extract obtained from the seeds of T. gardneriana (EETg), and from this, to evaluate its antioxidant activity through different methodologies, to investigate the bioaccessibility of EETg by determination of phenolic composition before and after in vitro digestion as well as to estimate its indirect bioavailability by chemical analysis of plasma and urine in rodents after oral administration. In general, EETg presented levels of antioxidant activity by DPPH, ABTS.+, FRAP and TBARS tests comparable to those described in specialized literature. The bioaccessibility indexes of phenolic compounds in EETg were 48.65 and 69.28% in the presence and absence of enzymes, respectively. Among the identified phenolics classes, flavonoids, represented by galloylated procyanidins, proved to be more bioaccessible (81.48 and 96.29% in the post-intestinal phase with and without enzymes, respectively). The oral administration in Wistar rats resulted in a significant decrease in plasma total antioxidant capacity (TAC) by FRAP assay 4 h after beginning the experiment. As to urine analysis, an increase in TAC by DPPH and FRAP was observed from 1 and 4 h after administration, respectively. UPLC-QTOF analysis of urine detected 2 metabolites originated from the degradation of phenolic compounds: hippuric acid and phenylacetil glycine. These results suggest that phenolic compounds in T. gardneriana, despite their antioxidant potential, are unstable under gastrointestinal conditions, being flavonoids the components with higher bioaccessibility; besides that, they showed limited bioavailability due to their rapid biotransformation and urinary elimination. / Triplaris gardneriana Wedd é uma espécie vegetal do semiárido brasileiro com sementes ricas em compostos fenólicos e alta capacidade antioxidante. O consumo de frutas e vegetais tem sido associado à baixa incidência de doenças crônicas causadas por estresse oxidativo. Dentre os componentes alimentares capazes de combater o excesso de radicais livres, os compostos fenólicos, de caráter antioxidante, têm sido bastante estudados.Contudo, a quantidade total destas substâncias presente nos alimentos não reflete necessariamente a porção bioativa absorvida e metabolizada pelo organismo. Neste contexto, o presente trabalho objetivou o desenvolvimento de um extrato etanólico obtido a partir das sementes de T. gardneriana(EETg),e a partir deste,caracterizar sua composição fenólica, avaliar sua atividade antioxidante através de diferentes metodologias, além deinvestigar a bioacessibilidade in vitro dos polifenóis presentes em EETg após digestão gastrointestinal simulada e estimar a biodisponibilidade indireta dos mesmos a partir da análise química do plasma e urina em roedores após sua administração oral. Em linhas gerais, EETg apresentou níveis de atividade antioxidante pelos ensaios DPPH, ABTS.+, FRAP e TBARS comparáveis àqueles descritos naliteratura especializada. Os índices de bioacessibilidade de compostos fenólicos em EETg foram de 48,65 e 69,28% na presença e ausência de enzimas, respectivamente. Dentre as classes fenólicas identificadas, os flavonoides, representados porprocianidinas acrescidas de grupos galoil, mostraram-se mais bioacessíveis (81,48 e 96,29% na fase pós-intestinal com e sem enzimas, respectivamente). A administração oral de EETg em ratos Wistar resultou em uma significativa diminuição da capacidade antioxidante total (CAT) do plasma pelo ensaio FRAP 4 h após o início do experimento. Para as amostras de urina, um aumento naCAT pelos testes DPPH e FRAP foi observado a partir de 1 e 4 h após a administração, respectivamente. Análises por UPLC-QTOF daurina detectou 2 metabólitos oriundos da degradação de compostos fenólicos: ácido hipúrico e fenilacetil glicina. Estes resultados sugerem que os compostos fenólicos deT. gardneriana, apesar do seu potencial antioxidante, são instáveis em condições gastrointestinais, sendo flavonoides os componentes com maior bioacessibilidade. Além disto, mostraram biodisponibilidade limitada devido à sua rápida biotransformação e eliminação urinária.

Le but de ce travail de thèse était de rechercher de nouvelles molécules inhibitrices de la SOD dans des extraits de substances naturelles. Cette enzyme majeure du stress oxydant étant impliquée dans de nombreux mécanismes de défense des cellules cancéreuses contre l'apoptose représente une voie thérapeutique d'avenir. Nous en avons recherché dans les champignons endophytes de plantes péruviennes, ces micro-organismes produisant de nombreux métabolites de défense des plantes hôtes. Ce travail de thèse a d'abord porté sur l'isolement, la culture, l'identification, l'extraction et la caractérisation des souches de champignons endophytes. Les analyses statistiques effectuées sur ces extraits avec les résultats obtenus en HPLC ont confirmé les problèmes de variabilités qualitative et quantitative pouvant être rencontrés au cours de la culture des endophytes et décrites dans la littérature. La seconde partie expérimentale a porté sur la recherche de furocoumarines dans les extraits obtenus, en raison de leur potentiel effet inhibiteur sur la SOD. Nous avons dérépliqué ces composés dans les extraits d'endophytes au cours de deux stratégies de spectrométrie de masse réalisées en mode d'ionisation négatif. La première approche, à l'aide d'un QTOF, a abouti à l'identification de deux furocoumarines, la 5-Methyl-4H-furo[2,3-b][1]benzopyran-4-one et la déhydropachyrrhizone. La seconde, à l'aide d'un OrbiTrap, a vu l'identification de quatre autres furocoumarines : l'Ochrocarpine A, la Moellendorffiline, l'Anisolactone et l'Anhydrorutarétine .Afin de compléter cette approche métabolomique et identifier les molécules inhibitrices de la SOD, nous avons tenté de mettre au point un test d'activité de cette enzyme, rapide, peu coûteux et réalisable en routine. Nous avons sélectionné le test au pyrogallol, mais celui-ci n'a pas démontré les qualités recherchées tant au niveau de la sensibilité que de la reproductibilité. Nous orientons à présent les recherches vers des approches différentes, la recherche directe d'adduits sur la SOD par LC-MS. / Superoxide dismutase is one of the major proteins controlling the oxidizing stress and cellular homeostasis. It is involved in numerous cancer cells proliferation processes. This protein is considered as major anti-cancer target for the development of new anti-cancer drugs. The goal of this work, was to research and identify an inhibitor of the SOD in endophytic fungi from Peruvian plants. These micro-organisms are known to produce numerous metabolites for host plants protection. During the preparation of endophytic extracts and their characterization by HPLC and statistical analyzes, we have pointed out a high quantitative and qualitative variability of the chemical content of endophytic extracts inside a same strain. Considering the literature we have focused our work on the identification of furocoumarins because of their potential inhibitory effect on the SOD. For this purpose two mass spectrometry strategies using negative ionization mode were carried out. With the QTOF mass spectrometer we have identified Methyl-4H-furo [2,3-b] [1] benzopyran-4-one and the déhydropachyrrhizone. With the OrbiTrap, the Ochrocarpine A, Moellendorffiline the Anisolactone and the Anhydrorutarétine were identified. Biological evaluation of the different extracts was performed using pyrogallol test. This investigation did not allow us to identify an inhibitor of the SOD. In the future we may consider seeking SOD inhibitors by looking at the formation SOD-chemical compound adducts using an LC-MS investigation.

The human gut microbiota plays a crucial role in human health and therefore imbalances in gut microbiota functioning can lead to the development of metabolic disorders. Short-chain fatty acids (SCFA), tricarboxylic acid (TCA) cycle metabolites, bile acids (BA) and other metabolites are the essential compounds for the metabolome. Per- and Polyfluoroalkyl substances (PFAS) are a group of chemicals that can be absorbed onto the lumen by food. Since many PFAS has shown to possess a long list of adverse effects on human health, the short term impact of PFAS on gut microbiota functioning using an in vitro model that mimics fermentation in the human colon was explored. Samples obtained from in vitro fecal fermentation were then analysed through high-performance liquid chromatography coupled to time-of-flight mass spectrometry (LC-qToF-MS) using targeted and non-targeted approaches. Fecal samples were donated by four donors which were put through a fermentation over 24 hours and treated with no PFAS mixture (control), low concentration mixture of PFAS (PFAS-L) and high concentration mixture of PFAS (PFAS-H). The PFAS mixture contained perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorohexanesulfonic acid (PFHxS), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA). For the fermentation, samples were collected after 0, 2, 4, 6, 8 and 24 hours. The samples were spun down and the supernatant was collected. Two approaches were applied to explore the metabolism in the fermentation extracts: The first one was a “targeted SCFA and TCA cycle metabolites analysis’’ and the second one was “untargeted analysis of polar and nonpolar metabolites’’. Results show that all TCA cycle metabolites displayed different trends with the compound and little to no variation between the treatments. However, although there was no significant difference, 4 core compounds of the TCA metabolism were lower after treatment with PFAS-L or PFAS-H. For the untargeted method, 78 of 2855 compounds were identified. When comparing control samples, PFAS-H and PFAS-L, 200 features showed statistically significant differences. Most of these had higher concentrations for PFAS-treated samples. When PFAS-H was compared only with PFAS-L, 30 features were found to be statistically significant, indicating that distinct concentrations of PFAS differentially affect gut microbiota metabolism. For future identification, further analysis must be done, preferably with MS/MS, to obtain more structural information for these significant metabolites, since the masses of the fraction ions are needed to narrow down the search in databases used to obtain the identity of an ion. PFAS and the concentration seem to have an impact on the gut microbiota, the study should be done with additional donors to archive trends applicable to a whole population.

O Brasil possui condições climáticas adequadas para o desenvolvimento de um grande número de frutas nativas e essa biodiversidade tem se tornado um caminho promissor para a descoberta de novos compostos bioativos capazes de ser utilizados na formulação de alimentos funcionais e medicamentos. Os compostos fenólicos apresentam ações específicas, podendo atuar como antioxidantes e anti-inflamatórios, assim prevenindo doenças crônicas. Diante do exposto, o objetivo deste trabalho foi avaliar o potencial antioxidante, antiinflamatório e a composição fenólica de dez frutas nativas brasileiras ainda pouco conhecidas: araçá-boi (Eugenia stipitata), cambuití-cipó (Sagerectia elegans), murici vermelho (Bysonima arthropoda), murici guassú (Byrsonima lancifolia), morango silvestre (Rubus rosaefolius), cambuci (Campomanesia phaea), jaracatiá-mamão (Jacaratia spinosa), juquirioba (Solanum alterno-pinatum), fruta-do-sabiá (Acnistus arborescens) e cajá (Spondias mombin L.). Os extratos etanólicos (80% v/v) das polpas foram analisados inicialmente quanto à capacidade de sequestro dos radicais ABTS&#8729+ e ROO&#8729. A atividade anti-inflamatória foi avaliada in vivo por meio do modelo de migração de neutrófilos induzida por carragenina, enquanto que a composição fenólica foi realizada por técnicas cromatográficas (CLAE-DAD e CG-EM). As 5 frutas com as maiores atividades biológicas foram ainda analisadas quanto à capacidade de sequestro de O2 &#8729-, HOCl e NO&#8729, atividade anti-inflamatória por meio do ensaio de ativação do fator nuclear-&kappa;B (NF-&kappa;B) e composição fenólica por espectrometria de massas de alta resolução (LC-ESI-QTOF-MS). Em relação ao sequestro do radical ABTS&#8729+ o cambuití-cipó apresentou a maior atividade (749,88 &mu;mol TE.g-1) e para o ROO&#8729 o murici vermelho apresentou a maior atividade antioxidante (559,09 &mu;mol TE.g-1). Os animais tratados com araçá-boi, cambuití-cipó, murici vermelho, cajá e morango silvestre apresentaram reduções no influxo de neutrófilos comparados ao grupo carragenina (p < 0,05). Por meio das técnicas de CLAE-DAD e CG-EM foi possível identificar compostos fenólicos pertencentes a classe dos flavonoides (catequina, epicatequina, rutina, quercetina glicosilada, kaempeferol glicosilado, quercetina, procianidina B1 e procianidina B2), sub-classe do ácido hidroxibenzóico (ácido gálico) e sub-classe dos ácidos hidrocinâmicos (ácido cumárico, ácido ferúlico e caféico). O araçá-boi, cambuití-cipó, murici vermelho, morango silvestre e cajá foram as cinco frutas que apresentaram as maiores atividades antioxidantes e/ou anti-inflamatórias, cujo perfil fenólico por LC-ESI-QTOF-MS indicou a presença de 18 compostos no araçá-boi, 32 no cambuitícipó, 26 no murici vermelho e 20 e 11 compostos no morango silvestre e cajá, respectivamente. Vários dos compostos fenólicos identificados foram encontrados pela primeira vez nessas espécies. O cambuiti-cipó e murici vermelho se destacaram em relação ao sequestro de HOCl (EC50 4,99 e 4,41 &mu;g mL-1, respectivamente) e o cambuití-cipó foi o mais ativo para desativar o radical O2 &#8729- (EC50 68,33 &mu;g mL-1) e NO&#8729 (EC50 0,78 &mu;g mL-1). Já os extratos de murici-vermelho, cambuití-cipó e morango silvestre inibiram significativamente a ativação do NF-&kappa;B. Portanto, as frutas nativas brasileiras são fontes de substâncias antioxidantes e anti-inflamatórias, bem como de uma grande diversidade de compostos fenólicos, os quais podem propiciar importantes benefícios para a saúde humana. / Brazil has favorable climatic conditions for the development of a large number of native fruits and this biodiversity has become a promising path towards the discovery of new bioactive compounds capable of being used in the formulation of functional foods and medicines. Phenolic compounds show specific action mechanisms, being able to act as antioxidants and anti-inflammatories, thus preventing chronic diseases. However, few Brazilian native fruits are well known and consumed by the population, undermining the investigation of chemical composition as well as the identification/quantification of bioactive compounds. In light of this, the objective of this work was to evaluate the antioxidant and anti-inflammatory potentials as well as the phenolic composition of ten underexploited Brazilian native fruits, namely: araçá-boi (Eugenia stipitata), cambuití-cipó (Sagerectia elegans), murici vermelho (Bysonima arthropoda), murici guassu (Byrsonima lancifolia), morango silvestre (Rubus rosaefolius), cambuci (Campomanesia phaea), jaracatiá-mamão (Jacaratia spinosa), juquirioba (Solanum alterno-pinatum), fruto-do-sabiá (Acnistus arborescens) and cajá (Spondias mombin L.). Pulps ethanolic extracts (80%, v/v) were initially analyzed regarding scavenging capacity of the ABTS&#8729+ and ROO&#8729 radicals. Antiinflammatory activity was evaluated in vivo using the carrageenan-induced neutrophil migration model, while phenolic composition was determined by chromatographic techniques (HPLC-PAD and GC-MS). The five fruits with the highest biological activities were analyzed for O2&#8729-, HOCl and NO&#8729 radicals scavenging capacities, for in vitro anti-inflammatory activity by nuclear factor-&kappa;B (NF-&kappa;B) activation, and for phenolic composition by High Resolution Mass Spectrometry (LC-ESI-QTOF-MS). In relation to ABTS&#8729+ radical scavenging cambuiticipó showed the highest activity (749.88 &mu;mol TE.g-1), while for ROO&#8729 scavenging, murici vermelho had the highest antioxidant activity (559.09 &mu;mol TE.g-1). The animals treated with araçá-boi, cambuití-cipó, murici vermelho, cajá and morango silvestre reported decreases in neutrophils influx compared to carrageenan group (p <0.05). It was possible to identify by HPLC-DAD and GC-MS techniques phenolic compounds belonging to the class of flavonoids (catechin, epicatechin, rutin, glycosylated quercetin, glycosylated kaempeferol, quercetin, procyanidin B1 and procyanidin B2), subclass of hydroxybenzoic acid (gallic acid) and subclass of hydrocinnamic acids (coumaric, ferulic and caffeic acids). Araçá-boi, cambuitícipó, murici vermelho, morango silvestre and cajá were the five fruits with the highest antioxidant and / or anti-inflammatory activities. The phenolic profile analysis by LC-ESIQTOF- MS pointed the presence of 18 compounds in araçá-boi, 32 in cambuití-cipó, 26 in murici vermelho, 20 in morango silvestre and 11 in cajá. Several of the identified phenolic compounds were found for the first time in these fruit species. Cambuiti-cipó and murici vermelho stood out in relation to HOCl scavenging (EC50 4.99 and 4.41 &mu;g.mL-1, respectively) and cambuití-cipó was the most active to deactivate both O2 &#8729- radical (EC50 68.33 &mu;g.mL-1) as NO&#8729 (EC 500.78 &mu;g.mL-1). Murici vermelho, cambuití-cipó and morango silvestre extracts significantly inhibited the activation of NF- &kappa;B.Therefore, Brazilian native fruits are sources of antioxidant and anti-inflammatory substances, as well as a great diversity of phenolic compounds, which can provide important benefits for human health.

As chamadas club drugs compreendem um vasto grupo de substâncias frequentemente utilizadas em bares, festas e raves, com a finalidade de intensificar o contato social e a estimulação sensorial. Englobam desde substâncias sintéticas comumente conhecidas, como a anfetamina, a metanfetamina, o MDMA, até moléculas de surgimento mais recente, denominadas novas substâncias psicoativas. Isoladas ou associadas a outras drogas, é possível que sejam causa de morte per se, ou que predisponham o usuário a envolver-se em situações potencialmente fatais, sendo necessário que os órgãos de Perícia Criminal (Institutos Médico Legais e Institutos de Criminalística) estejam aptos a detectar e quantificar essas substâncias em amostras biológicas. O presente trabalho teve como objetivo desenvolver um método analítico para identificação e quantificação de club drugs em sangue total, utilizando cromatografia líquida acoplada a espectrometria de massas com analisador híbrido quadrupolotempo de voo (LC-QTOF). Após o desenvolvimento do método, este foi validado utilizando as diretrizes do guia de validação do Scientific Working Group for Forensic Toxicology (SWGTOX), sendo analisados de linearidade, limite de detecção, limite de quantificação, efeito matriz, precisão intradia, precisão interdia, exatidão e integridade de diluição, além de recuperação e eficiência do processo. O método desenvolvido compreendeu a determinação de MDA, MDMA, 2C-B, DOB, cetamina, mCPP, cocaína e cocaetileno. Amostras provenientes de casos reais de morte não natural, oriundas do Instituto Médico Legal Aristoclides Teixeira de Goiânia - GO foram analisadas pelo método desenvolvido. 56 casos foram selecionados, em sua maioria com histórico de morte por projétil de arma de fogo e acidente de transito. Das 56 amostras analisadas, 28,5% (n=16) foram positivas para cocaína e/ou cocaetileno. As demais substâncias pesquisadas não foram encontradas nas amostras. / Club drugs are a large group of substances consumed in pubs, parties and raves, aiming to intensify social contact and sensorial stimulation. The term comprises largely known substances such as amphetamine, methamphetamine, 3,4-methylenodioxymethamphetamine (MDMA), as well as so-called new psychoactive substances, which are synthetic drugs recently developed or recently introduced in drug market. Club drugs can be taken alone, combined with each other or, most frequently, with alcohol or other commonly abused drugs such as cocaine. In any of these situations, club drugs can possibly be the cause of death or potentialize the involvement of the user with crime and potentially fatal behavior. Thus, official organisms in charge of criminal investigation must be capable of identifying and quantifying these substances in biological samples. The present work aimed the development of an analytical method to identify and quantify club drugs in whole blood, using liquid chromatography - mass spectrometry with hybrid analyzer quadrupole - time of flight (LC-QTOF). After analytical development, the method was validated according to do Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, evaluating linearity, limit of detection, limit of quantification, matrix effect, precision, intermediate precision, bias and dilution integrity, besides recovery and process efficiency. The developed method comprised MDA, MDMA, 2C-B, DOB, ketamine, mCPP, cocaine and cocaethylene determination. Real samples related to non-natural deaths were collected at Institute of the Legal Medicine Aristoclides Teixeira, Goiânia, Goiás, Brazil, and analyzed by the developed method. 56 cases were selected, most of them related to fire gun injury and traffic events, 28,5% (n=16) of them being positive for cocaine and/or cocaethylene. None of the other drugs comprised in the analysis were detected in these samples.

As chamadas club drugs compreendem um vasto grupo de substâncias frequentemente utilizadas em bares, festas e raves, com a finalidade de intensificar o contato social e a estimulação sensorial. Englobam desde substâncias sintéticas comumente conhecidas, como a anfetamina, a metanfetamina, o MDMA, até moléculas de surgimento mais recente, denominadas novas substâncias psicoativas. Isoladas ou associadas a outras drogas, é possível que sejam causa de morte per se, ou que predisponham o usuário a envolver-se em situações potencialmente fatais, sendo necessário que os órgãos de Perícia Criminal (Institutos Médico Legais e Institutos de Criminalística) estejam aptos a detectar e quantificar essas substâncias em amostras biológicas. O presente trabalho teve como objetivo desenvolver um método analítico para identificação e quantificação de club drugs em sangue total, utilizando cromatografia líquida acoplada a espectrometria de massas com analisador híbrido quadrupolotempo de voo (LC-QTOF). Após o desenvolvimento do método, este foi validado utilizando as diretrizes do guia de validação do Scientific Working Group for Forensic Toxicology (SWGTOX), sendo analisados de linearidade, limite de detecção, limite de quantificação, efeito matriz, precisão intradia, precisão interdia, exatidão e integridade de diluição, além de recuperação e eficiência do processo. O método desenvolvido compreendeu a determinação de MDA, MDMA, 2C-B, DOB, cetamina, mCPP, cocaína e cocaetileno. Amostras provenientes de casos reais de morte não natural, oriundas do Instituto Médico Legal Aristoclides Teixeira de Goiânia - GO foram analisadas pelo método desenvolvido. 56 casos foram selecionados, em sua maioria com histórico de morte por projétil de arma de fogo e acidente de transito. Das 56 amostras analisadas, 28,5% (n=16) foram positivas para cocaína e/ou cocaetileno. As demais substâncias pesquisadas não foram encontradas nas amostras. / Club drugs are a large group of substances consumed in pubs, parties and raves, aiming to intensify social contact and sensorial stimulation. The term comprises largely known substances such as amphetamine, methamphetamine, 3,4-methylenodioxymethamphetamine (MDMA), as well as so-called new psychoactive substances, which are synthetic drugs recently developed or recently introduced in drug market. Club drugs can be taken alone, combined with each other or, most frequently, with alcohol or other commonly abused drugs such as cocaine. In any of these situations, club drugs can possibly be the cause of death or potentialize the involvement of the user with crime and potentially fatal behavior. Thus, official organisms in charge of criminal investigation must be capable of identifying and quantifying these substances in biological samples. The present work aimed the development of an analytical method to identify and quantify club drugs in whole blood, using liquid chromatography - mass spectrometry with hybrid analyzer quadrupole - time of flight (LC-QTOF). After analytical development, the method was validated according to do Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, evaluating linearity, limit of detection, limit of quantification, matrix effect, precision, intermediate precision, bias and dilution integrity, besides recovery and process efficiency. The developed method comprised MDA, MDMA, 2C-B, DOB, ketamine, mCPP, cocaine and cocaethylene determination. Real samples related to non-natural deaths were collected at Institute of the Legal Medicine Aristoclides Teixeira, Goiânia, Goiás, Brazil, and analyzed by the developed method. 56 cases were selected, most of them related to fire gun injury and traffic events, 28,5% (n=16) of them being positive for cocaine and/or cocaethylene. None of the other drugs comprised in the analysis were detected in these samples.

O Brasil possui condições climáticas adequadas para o desenvolvimento de um grande número de frutas nativas e essa biodiversidade tem se tornado um caminho promissor para a descoberta de novos compostos bioativos capazes de ser utilizados na formulação de alimentos funcionais e medicamentos. Os compostos fenólicos apresentam ações específicas, podendo atuar como antioxidantes e anti-inflamatórios, assim prevenindo doenças crônicas. Diante do exposto, o objetivo deste trabalho foi avaliar o potencial antioxidante, antiinflamatório e a composição fenólica de dez frutas nativas brasileiras ainda pouco conhecidas: araçá-boi (Eugenia stipitata), cambuití-cipó (Sagerectia elegans), murici vermelho (Bysonima arthropoda), murici guassú (Byrsonima lancifolia), morango silvestre (Rubus rosaefolius), cambuci (Campomanesia phaea), jaracatiá-mamão (Jacaratia spinosa), juquirioba (Solanum alterno-pinatum), fruta-do-sabiá (Acnistus arborescens) e cajá (Spondias mombin L.). Os extratos etanólicos (80% v/v) das polpas foram analisados inicialmente quanto à capacidade de sequestro dos radicais ABTS&#8729+ e ROO&#8729. A atividade anti-inflamatória foi avaliada in vivo por meio do modelo de migração de neutrófilos induzida por carragenina, enquanto que a composição fenólica foi realizada por técnicas cromatográficas (CLAE-DAD e CG-EM). As 5 frutas com as maiores atividades biológicas foram ainda analisadas quanto à capacidade de sequestro de O2 &#8729-, HOCl e NO&#8729, atividade anti-inflamatória por meio do ensaio de ativação do fator nuclear-&kappa;B (NF-&kappa;B) e composição fenólica por espectrometria de massas de alta resolução (LC-ESI-QTOF-MS). Em relação ao sequestro do radical ABTS&#8729+ o cambuití-cipó apresentou a maior atividade (749,88 &mu;mol TE.g-1) e para o ROO&#8729 o murici vermelho apresentou a maior atividade antioxidante (559,09 &mu;mol TE.g-1). Os animais tratados com araçá-boi, cambuití-cipó, murici vermelho, cajá e morango silvestre apresentaram reduções no influxo de neutrófilos comparados ao grupo carragenina (p < 0,05). Por meio das técnicas de CLAE-DAD e CG-EM foi possível identificar compostos fenólicos pertencentes a classe dos flavonoides (catequina, epicatequina, rutina, quercetina glicosilada, kaempeferol glicosilado, quercetina, procianidina B1 e procianidina B2), sub-classe do ácido hidroxibenzóico (ácido gálico) e sub-classe dos ácidos hidrocinâmicos (ácido cumárico, ácido ferúlico e caféico). O araçá-boi, cambuití-cipó, murici vermelho, morango silvestre e cajá foram as cinco frutas que apresentaram as maiores atividades antioxidantes e/ou anti-inflamatórias, cujo perfil fenólico por LC-ESI-QTOF-MS indicou a presença de 18 compostos no araçá-boi, 32 no cambuitícipó, 26 no murici vermelho e 20 e 11 compostos no morango silvestre e cajá, respectivamente. Vários dos compostos fenólicos identificados foram encontrados pela primeira vez nessas espécies. O cambuiti-cipó e murici vermelho se destacaram em relação ao sequestro de HOCl (EC50 4,99 e 4,41 &mu;g mL-1, respectivamente) e o cambuití-cipó foi o mais ativo para desativar o radical O2 &#8729- (EC50 68,33 &mu;g mL-1) e NO&#8729 (EC50 0,78 &mu;g mL-1). Já os extratos de murici-vermelho, cambuití-cipó e morango silvestre inibiram significativamente a ativação do NF-&kappa;B. Portanto, as frutas nativas brasileiras são fontes de substâncias antioxidantes e anti-inflamatórias, bem como de uma grande diversidade de compostos fenólicos, os quais podem propiciar importantes benefícios para a saúde humana. / Brazil has favorable climatic conditions for the development of a large number of native fruits and this biodiversity has become a promising path towards the discovery of new bioactive compounds capable of being used in the formulation of functional foods and medicines. Phenolic compounds show specific action mechanisms, being able to act as antioxidants and anti-inflammatories, thus preventing chronic diseases. However, few Brazilian native fruits are well known and consumed by the population, undermining the investigation of chemical composition as well as the identification/quantification of bioactive compounds. In light of this, the objective of this work was to evaluate the antioxidant and anti-inflammatory potentials as well as the phenolic composition of ten underexploited Brazilian native fruits, namely: araçá-boi (Eugenia stipitata), cambuití-cipó (Sagerectia elegans), murici vermelho (Bysonima arthropoda), murici guassu (Byrsonima lancifolia), morango silvestre (Rubus rosaefolius), cambuci (Campomanesia phaea), jaracatiá-mamão (Jacaratia spinosa), juquirioba (Solanum alterno-pinatum), fruto-do-sabiá (Acnistus arborescens) and cajá (Spondias mombin L.). Pulps ethanolic extracts (80%, v/v) were initially analyzed regarding scavenging capacity of the ABTS&#8729+ and ROO&#8729 radicals. Antiinflammatory activity was evaluated in vivo using the carrageenan-induced neutrophil migration model, while phenolic composition was determined by chromatographic techniques (HPLC-PAD and GC-MS). The five fruits with the highest biological activities were analyzed for O2&#8729-, HOCl and NO&#8729 radicals scavenging capacities, for in vitro anti-inflammatory activity by nuclear factor-&kappa;B (NF-&kappa;B) activation, and for phenolic composition by High Resolution Mass Spectrometry (LC-ESI-QTOF-MS). In relation to ABTS&#8729+ radical scavenging cambuiticipó showed the highest activity (749.88 &mu;mol TE.g-1), while for ROO&#8729 scavenging, murici vermelho had the highest antioxidant activity (559.09 &mu;mol TE.g-1). The animals treated with araçá-boi, cambuití-cipó, murici vermelho, cajá and morango silvestre reported decreases in neutrophils influx compared to carrageenan group (p <0.05). It was possible to identify by HPLC-DAD and GC-MS techniques phenolic compounds belonging to the class of flavonoids (catechin, epicatechin, rutin, glycosylated quercetin, glycosylated kaempeferol, quercetin, procyanidin B1 and procyanidin B2), subclass of hydroxybenzoic acid (gallic acid) and subclass of hydrocinnamic acids (coumaric, ferulic and caffeic acids). Araçá-boi, cambuitícipó, murici vermelho, morango silvestre and cajá were the five fruits with the highest antioxidant and / or anti-inflammatory activities. The phenolic profile analysis by LC-ESIQTOF- MS pointed the presence of 18 compounds in araçá-boi, 32 in cambuití-cipó, 26 in murici vermelho, 20 in morango silvestre and 11 in cajá. Several of the identified phenolic compounds were found for the first time in these fruit species. Cambuiti-cipó and murici vermelho stood out in relation to HOCl scavenging (EC50 4.99 and 4.41 &mu;g.mL-1, respectively) and cambuití-cipó was the most active to deactivate both O2 &#8729- radical (EC50 68.33 &mu;g.mL-1) as NO&#8729 (EC 500.78 &mu;g.mL-1). Murici vermelho, cambuití-cipó and morango silvestre extracts significantly inhibited the activation of NF- &kappa;B.Therefore, Brazilian native fruits are sources of antioxidant and anti-inflammatory substances, as well as a great diversity of phenolic compounds, which can provide important benefits for human health.

Il est maintenant avéré que les contaminants présents dans les milieux aquatiques peuvent être toxiques à l’état de traces voire d’ultra traces. Il est donc important de développer des méthodes d’analyse performantes et sensibles pouvant atteindre ces niveaux de concentration. Dans cette optique, les techniques chromatographiques couplées à la spectrométrie de masse (GC-MS et LC-MS/MS) sont généralement utilisées pour l’analyse des polluants organiques. Les composés présents dans les milieux aquatiques possèdent des propriétés physico-chimiques très variées, d’apolaires à très polaires. Ainsi, développer une analyse simultanée pour toutes ces molécules représente un challenge analytique. Dans ce travail de thèse, plusieurs étapes du processus analytique ont été évaluées : la préparation de l’échantillon par extraction sur phase solide (SPE), la séparation par chromatographie et la détection par spectrométrie de masse. Une liste de composés modèles couramment retrouvés dans les milieux aquatiques a été établie pour conduire ces essais. Les méthodes d’extraction en phase solide hors ligne et en ligne ont été développées dans une optique d’analyse multirésidus à un niveau de traces. Les méthodes chromatographiques, gazeuse et liquide, couplées à la spectrométrie de masse ont été étudiées pour favoriser une analyse exhaustive et sensible. La dernière partie a consisté à appliquer les méthodes développées pour une approche d’analyse non ciblée. / It is now widely recognized that contaminants present in aquatic environments can be toxic at traces or even ultra-traces level. Therefore, it is important to develop efficient and sensitive analytical methods to reach these levels of concentration. In that respect, chromatographic techniques coupled to mass spectrometry (GC-MS and LC-MS / MS) are commonly used for the analysis of organic pollutants. The substances encountered in aquatic environments display a large range of physico-chemical properties, from apolar to very polar. Thus, developing a simultaneous analysis for all these molecules represents an analytical challenge. In this pHD work, several steps of the analytical process have been investigated: sample preparation by solid phase extraction (SPE), chromatographic separation and the detection by mass spectrometry. A list of model compounds commonly determined in aquatic environments was established to conduct the tests. Solid phase extraction methods, offline and online, were developed in a multiresidue analysis aim at traces level. Chromatographic methods, gaseous and liquid, coupled to mass spectrometry were studied to obtain an exhaustive and sensitive analysis. The last part consisted to apply the developed methods for a non-targeted analysis approach.

Dentre as espÃcies existentes de maracujÃ, somente trÃs sÃo utilizadas pela indÃstria e, entre estas, exclusivamente a Passiflora edulis atende ao mercado de alimentos, pois alÃm desta espÃcie possuir atributos sensoriais bastante atrativos, ainda possui diversos benefÃcios para melhoria da saÃde. Entretanto, nem sempre as caracterÃsticas da fruta in natura sÃo mantidas apÃs serem processadas. Sendo assim, este estudo tem por objetivo avaliar o perfil de metabÃlitos presentes em suco de maracujà apÃs o tratamento tÃrmico de pasteurizaÃÃo e de esterilizaÃÃo em diferentes condiÃÃes de tempo e temperatura e ainda avaliar o efeito do tratamento tÃrmico no suco utilizando-se processamento com ultrassom. As variÃveis utilizadas no processamento tÃrmico foram tempo de retenÃÃo (0, 4, 15, 30 e 60 segundos), temperatura (85 e 140ÂC) e para o processo nÃo tÃrmico com ultrassom de ponteira foram variadas as potÃncias (50.000, 75.000 e 100.000 W/L) e o tempo (1; 2,5; e 5 min). Para extraÃÃo dos compostos utilizou-se metanol e foi feita a identificaÃÃo das amostras em UPLC-MS. TambÃm foram realizadas anÃlises de ressonÃncia magnÃtica (RMN) e PolifenÃis ExtraÃveis Totais (PET). Para o tratamento de dados foi feita anÃlise do componente principal (PCA) para o UPLC-MS e RMN. Os resultados foram comparados a uma amostra controle, tendo sido encontrados 27 picos, dos quais 4 foram degradados, 7 foram formados e 16 permaneceram inalterados para o tratamento tÃrmico e para o ultrassom de ponteira nÃo houve grandes variaÃÃes, quanto ao teor de polifenÃis, estes tornaram-se disponÃveis com aumento da temperatura, bem como com potÃncia mais alta por um tempo de 5 minutos. O processo tÃrmico pode ser controlado a partir de dados obtidos sobre o perfil de suco de maracujà submetido a tratamento tÃrmico. / Brazil has a great biodiversity, which should be explored and transformed into food and products to be consumed. Among existing species of passion fruit, only three are used by industry and among these, exclusively the Passiflora edulis serves the food market, because this species has very attractive sensory attributes,also is related to several benefits to improvhealth. However, the fruit characteristics âin naturaâ are not always maintainedthem after processing. Thus, this study aims to evaluate the metabolic profile present in passion fruit juice after heat treatment of sterilization further morepasteurization in different conditions of time and temperature and still submit the juice without heat treatment to a ultrasoundprocessing. The studied variables in heat processing wereretentiontime (0, 4, 15, 30 and 60 seconds), temperature (85 and 140 ÂC) and the non-thermal ultrasound process withpower (50.000, 75.000 e 100.000 W/L) and time (1, 2.5 and 5 minutes). For the extraction of the compounds were used methanol, and sample identification performed in LC-MS. Also was carried out Nuclear Magnetic Resonance analysis (NMR) andTotal Extractable Polyphenols (TEP). In the methodology employed for processing with ultrasound, the samples were subjected to different power and time. Then, was performed the same reading and extracting methodology used for heat processing. For treatment of the data was performed Principal Component Analysis (PCA) for LC-MS and NMR, the polyphenols were analyzed by the method described by Singleton and Rossi (1965), with adaptations. The results were compared to a control sample (no treatment). Were found 32 chromatographic peaks, of which 4 were degraded, 12 were formed and 16 remained unchanged. With increasing temperature, there was degradation of phenolic compounds. The heat process can be controlled from data obtained on the passion fruit juice profile submitted to heat treatment.