Per- and polyfluoroalkyl substances (PFAS) encompass a large group of manufactured compounds that have been used in various production processes such as food packaging, commercial products, workplaces, homes, water supplies, and food. PFAS are persistent, resistant to degradation, and can bioaccumulate. Although an exposure limit that predicts adverse health effects has yet to be determined, the Center for Disease Control and Prevention’s 2015-16 health survey found average blood levels of 4.72 ng/ml for PFOS and 1.56 ng/ml for PFOA. The objective of this research was to evaluate the use of laminar flow tandem mass spectrometry following solid phase extraction (SPE) using weak anion exchange (WAX) properties on the detection and quantitation of PFAS compounds. Seven-point calibration standards applied to this research were prepared using certified reference materials (Wellington Laboratories, Ontario, CA), and calibrators were run without sample extraction. The concentrations varied slightly based on the PFAS analyte of interest. All samples and quality controls were prepared by spiking certified reference material (Wellington Laboratories) into pooled human serum (BioIVT, Westbury, NY, USA). A laminar flow QSight®220 ultra-high pressure liquid chromatography-tandem mass spectrometer (LC-MS/MS, PerkinElmer, Waltham, MA, USA) was equipped with a Selectra C18 100 x 2.1mm x 3μm (UCT, Bristol, PA, USA) column with a Brownlee C18 delay column (PerkinElmer) and followed the LC-MS/MS parameters developed for the method. Extraction was accomplished using a WAX SPE column (UCT, ECWAX053) by first conditioning the columns with 1 mL of methanol (Fisher Scientific, Fair Lawn, NJ, USA) followed by 1 mL of 100 mM pH 7 phosphate buffer (Acros Organics, Geel, Belgium, EU). Samples were loaded onto the column at a rate of 1-2 mL/min. The SPE cartridges were washed with 1 mL of 100 mM pH 7 phosphate buffer and 1 mL of millipore water (Millipore Milli- Q Ultrapure Type 1 water system, Millipore Sigma, Burlington, MA, USA), then dried under full flow for 5 minutes. Elution was carried out with 2.5mL of a 98:2 methanol: OptimaTM grade ammonium hydroxide (Fisher Scientific) solution. The eluted samples were then evaporated to dryness using a MULTIVAP® Nitrogen Evaporator (Organomation,Berlin,MA,USA) at 55°C and 5psi. All samples were reconstituted in 100 μL of a 96:4 methanol:water solution. The parameters assessed followed Academy Standards Board Standard 036: Standard Practices for Method Validation in Forensic Toxicology, including matrix interferences, limit of detection (LOD), limit of quantitation (LOQ), a recovery study, and a calibration model. The results of the study were gathered from the following eleven analytes: PFBA, PFBS, PFHxA, PFHpA, PFHxS, PFOA, PFOS, PFNA, PFDA, PFUnA, and PFDoA. Depending on the analyte, a lower LOQ was established at 0.16 – 1.75ng/mL and an upper LOQ at 43.75 – 51.41 ng/mL. Based on the established linear calibration model an LOD in the range of 0.11 - 0.51 ng/mL was achieved. All eleven PFAS analytes showed an acceptable bias of ±20%. All analytes showed a between-run precision (%CV) in an acceptable range of ±20%. No matrix interferences were detected. The average recovery for SPE ranges from 77.64- 104.73% with recovery of 77.64% for PFBS, 83.89% for PFBA, and 95.64-104.73% for PFHxA, PFHpA, PFHxS, PFOA, PFOS, PFNA, PFDA, PFUnA, and PFDoA. Utilizing the UCT WAX SPE column, good recovery for the PFAS compounds was demonstrated. Further, the extraction technique was efficient for high throughput analysis with the extraction time comparable to other traditional SPE methods. The total analytical run time of 11 minutes using the QSight®220 coupled with the UCT Selectra C18 100 x 2.1mm x 3μm column allowed for adequate re-equilibration and system washes to prevent carryover and contamination of these persistent pollutants with excellent chromatography. Having the ability to efficiently and accurately quantify PFAS compounds in biological matrices will allow for better understanding of prevalence, bioaccumulation in biological matrices, and will aid in understanding how these concentrations relate to various health outcomes.

An acetonitrile based protein extraction method was developed that demonstrated high efficient and effective removal of high abundant proteins from both human and murine serum. The protein content of the extract was characterised using gel electrophoresis, the Bradford assay and liquid chromatography tandem mass spectrometry (LC-MS/MS) with database searching. Selected reaction monitoring (SRM) analysis was used to quantify the levels of high abundant serum proteins to further validate the extraction methodology. The ACN depletion method, in combination with artificial neural networks (ANNs) data mining software, was applied to a murine growth hormone (GH) gene doping study with the aim of identifying biomarker ions capable of detecting gene doping. The LC-MS and ANNs analysis approach failed to conclusively identify a biomarker to gene doping in the mouse model. However, the application of the same technique to serum from a rhGH administration study in humans, returned models capable of discriminating between rhGH treated placebo states. The ion identified as being the most discriminatory was characterised using mass spectrometry, and was derived from the protein leucine-rich a-2-glycoprotein (LRG). Multiple LRG related tryptic peptides were identified as being up-regulated upon dosing with recombinant human GH (rhGH). A high throughput LC-MS/MS and SRM approach was developed to quantify proteins in human serum. The approach was validated by comparison of LC-MS/MS derived APO A1 concentrations with those obtained using established clinical analyser technologies. The LC-MS/MS methodology was applied to a large cohort of 257 serum samples from two rhGH administration studies performed at Royal Free Hospital . The two administrations included serum samples from 15 individuals who had been dosed daily with rhGH. Serum concentrations of the established rhGH biomarker insulin-like growth factor-I (IGF-I) were quantified by LC-MS/MS and compared well with those determined using two different immunoassay-based methodologies. Serum concentrations of the LRG protein were measured simultaneously with IGF-I and appeared to increase in 14 of the 15 rhGH dosed individuals. Combining the LRG and IGF-I data further increased the separation of rhGH treated and placebo states within each individual, and the application of ANNs analysis showed that the combination of the two proteins increased the discrimination characteristics over using IGF-I alone. The murine equivalent of the LRG protein was identified and SRM transitions for a tryptically derived peptide were developed, along with transitions for monitoring a peptide from the murine IGF-I protein. These transitions were used to quantify the two proteins in the remaining aliquots from a murine GH gene doping experiment, however neither protein appeared to increase in the GH +ve plasmid samples that were analysed.

In recent years, the indiscriminate use of pesticides and a lack of adoption of good agricultural practices have been evidenced by the results of analyzes of residues of pesticides in food. Thus, the goal of this work was to develop and validate two methods for determination of pesticide residues in plum, apple, pear and peach, with analysis by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). In the development of the QuEChERS method, different conditions of the extract clean-up were tested. In the validation results, the linear range of the method was 5 to 200 μg kg-1 with recoveries were between 70 and 120% and RSD ≤20%. The practical limit of quantification (LOQ) for 161 compounds was 10 μg kg-1. In MSPD optimization, different sorbents (C18, florisil®, PSA, alumina, amino, silica, chitosan, Oasis® HLB, StrataTM-X and fluoridated) were tested as solid support in different proportions with the sample. The homogenization was carried out with metallic spheres and extraction with acetonitrile and ultrasound. Accuracy and precision results obtained by MSPD were adequate for 131 pesticides residues studied, with recovery results ranging between 70 and 120%, with RSD ≤ 20%. The studied compounds presented practical LOQ of 10 μg kg-1. In the analysis of 100 samples of the studied fruits, different pesticides residues were found in concentrations ranging from <LOQ to 2.334,6 μg kg-1. / Nos últimos anos, o uso indiscriminado de agrotóxicos e a não adoção das boas práticas agrícolas têm sido evidenciados pelos resultados de análises de resíduos de agrotóxicos em alimentos. Assim, este trabalho teve como objetivo desenvolver e validar dois métodos para determinação de resíduos de agrotóxicos em ameixa, maçã, pera e pêssego, com análise por cromatografia líquida de ultra eficiência acoplada à espectrometria de massas em série (UHPLC-MS/MS). No desenvolvimento do método QuEChERS, diferentes condições da limpeza do extrato foram testadas. Nos resultados de validação, o intervalo linear do método foi de 5 a 200 μg kg-1, com recuperações entre 70 e 120% e RSD ≤ 20%. O limite de quantificação prático (LOQ) para 161 compostos foi de 10 μg kg-1. No desenvolvimento da técnica MSPD foram testados diferentes sorventes (C18, florisil®, PSA, alumina, amino, sílica, quitosana, Oasis® HLB, StrataTM-X e fase fluorada) como suporte sólido em diferentes proporções com a amostra. A homogeneização foi realizada com auxílio de esferas metálicas e a extração com acetonitrila e ultrassom. Os resultados de exatidão e precisão obtidos por MSPD foram adequados para resíduos de 131 agrotóxicos, com resultados de recuperação entre 70 e 120%, with RSD ≤ 20%. Os compostos estudados apresentaram LOQ prático de 10 μg kg-1. Na análise de 100 amostras das frutas estudadas, foram quantificados resíduos de diferentes agrotóxicos em concentrações entre <LOQ e 2.334,6 μg kg-1.

The aim of the thesis was to analyze the historical pharmaceutical preparations, including the determination of the active substance and identify theirs possible degradation products. A historical pharmaceutical preparation of naloxone was analyzed by mass spectrometry. Historical pharmaceutical preparations of adrenaline and ephedrine were analyzed by UHPLC-MS and were quantified using a calibration curve. In the historical injection solution of naloxone, "NARCAN", dated around 1980, there were no significant degradation products and the measured mass and UV spectrum was consistent with the spectrum of naloxone. The analyzed sample of naloxone was stable even after 35 years of storage. In the analyzed historical injection solution of adrenaline, "Adrenalin Hydrochlor., Dr. Heisler" (dated between 1917 and 1938) was determined 5.26 ± 0.11 % of the declared amount of adrenaline. In the measured spectras were noticeable degradation products, which have not been described in the literature yet and their identification was beyond the scope of this paper. The analyzed sample of adrenaline was almost completely degraded during about ninety years. The stability test carried out with four standard solutions of adrenaline proved influence of oxygen, light, temperature and time on the degradation of adrenaline. In...

Occurrence, accumulation and subsequent fate of pharmaceuticals in environment currently represent a very actual topic. Worldwide, thousands of tons of pharmaceutical substances are consumed every year. A large portion of pharmaceuticals is, in unchanged or metabolized forms, disposed via sewage systems and wastewater treatment plants. Considering the fact that wastewater treatment processes are not able to completely eliminate all active substances or their metabolites, pharmaceuticals are systematically washing out into the water system and increasingly contaminate the ground and surface waters. The problematics of continuous control and progressive elimination of pharmaceutical residues from environment are still not completely solved. Thus, the development and availability of accurate and fast commercial analyses are highly desired. The aim of this diploma thesis was the optimization and validation of multi-residue UHPLC- MS/MS analytical method designated for the determination of 52 pharmaceuticals in drinking and waste waters. The work was carried out in laboratories of ALS Czech Republic. An analytical method was subsequently used for monitoring of pharmaceuticals in both drinking and waste waters, as well as for the determination of efficiency of removing these compounds within the...

O maracujá, nome popular atribuído ao fruto das diversas espécies do gênero Passiflora, da família Passifloraceae, é amplamente comercializado e consumido no mundo, sendo o Brasil um dos maiores produtores do fruto. Alguns estudos apontam possível toxicidade relacionada às espécies de Passiflora, principalmente P. incarnata. No entanto, há pouco conhecimento acerca das espécies P. edulis e P. alata, sobretudo em relação à polpa e sementes. Os extratos da polpa e das sementes dos frutos dessas duas espécies de \"maracujá\", Passiflora alata e Passiflora edulis, foram estudados com o objetivo de identificar alcaloides harmânicos, pelo preparo das amostras por extração por sorção em barra magnética recoberta com polidimetilsiloxano (SBSE-PDMS) e SBSE recoberta com polietilenoglicol silicone (SBSE-EG Silicone) e análise por cromatografia líquida de alta eficiência com detector por fluorescência (CLAE-Flu) e cromatografia líquida de ultra eficiência acoplada à espectrometria de massas sequencial (CLUE-EM/EM). A análise dos alcaloides harmana e harmina nos extratos da polpa de P. alata foi feita por meio do método de adição de padrão e mostrou menor quantidade destes alcaloides, em comparação com os resultados da análise dos extratos da polpa dos frutos de P. edulis, no trabalho de Pereira e colaboradores. As análises CLUE-EM e CLUE-EM/EM possibilitaram a identificação dos alcaloides nos extratos: nas sementes de P. alata, os alcaloides harmana, harmina, harmol, harmalol e harmalina foram identificados, enquanto que na polpa, harmana e harmina tiveram a confirmação da sua presença. Nos extratos da polpa dos frutos de P. edulis observou-se os alcaloides harmana, harmina e harmalina. E nas sementes de P. edulis harmina foi encontrada, porém há indícios da presença de harmana. A literatura sobre as barras de SBSE-EG Silicone Twister&®; não relata nenhum estudo relacionado ao seu uso para extração e concentração de alcaloides harmânicos nos extratos de P. alata e P. edulis. Por isso foi proposto inicialmente a aplicação do planejamento fatorial fracionário para otimização do método de extração, utilizando os padrões comerciais dos alcaloides harmana e harmina. O planejamento experimental revelou as variáveis principais e seus níveis de importância, e a partir destes resultados foi realizado o estudo cinético dos tempos de extração e de dessorção das barras de SBSE-EG Silicone. Porém, os resultados mostraram que as barras de SBSE-EG Silicone não são adequadas para a extração dos alcaloides harmana e harmina, uma vez que a recuperação obtida foi baixa, na ordem de 30%. / \"Maracujá\" is the popular name given to the fruit of several species of Passiflora genus, from Passifloraceae family, it is widely commercialized and consumed around the world, and Brazil is one of the largest producers of this fruit. Some studies pointed out the possible toxicity related to Passiflora species, mainly P. incarnata. Although, there is a lack of knowledge about the P. edulis and P. alata species, especially with regards to the pulp and seeds. The extracts of pulp and seeds from the \"maracujá\" species Passiflora alata and Passiflora edulis, were studied in order to identify harman alkaloids. The samples were prepared by extraction with sorptive stir bar coated with polydimethylsiloxane (SBSE-PDMS) and SBSE coated with polyethylene glycol silicon (SBSE-EG Silicone). The samples were analyzed by high performance liquid chromatography with fluorescence detector (HPLC-Flu), and ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Harmane and harmine alkaloids in P. alata pulp extracts were analyzed using the standard addition method and the results showed a lower amount of these alkaloids, compared with the test results for the extracts from the P. edulis pulp in the work of Pereira et al. UHPLC-MS and UHPLC-MS/MS analysis enabled to identify the alkaloids amount present in the extracts. In the P. alata seeds extract the following alkaloids were identified harmane, harmine, harmol, harmalol and harmaline, while in the pulp extract, harmane and harmine were confirmed. In the extracts of P. edulis pulp the alkaloids identified were harmane, harmine and harmaline. And in the P. edulis seeds extract the harmine alkaloid was found, some indications of the presence of harmana were observed. The literature about SBSE-EG Silicone Twister® bars reports no study related to their use for extraction and concentration of harman alkaloids in P. alata and P. edulis. Thus, it was initially proposed the application of fractional factorial design to optimize the extraction method using commercial standards of harmane and harmine alkaloids. The experimental design revealed the main variables and their importance levels, and from these results kinetic studies were performed for the extraction and desorption times of SBSE-EG Silicone bars. However, the results showed SBSE-EG Silicone bars are not suitable for the extraction of harmane and harmine alkaloids, since the recovery obtained was low, on the order of 30%.

O maracujá, nome popular atribuído ao fruto das diversas espécies do gênero Passiflora, da família Passifloraceae, é amplamente comercializado e consumido no mundo, sendo o Brasil um dos maiores produtores do fruto. Alguns estudos apontam possível toxicidade relacionada às espécies de Passiflora, principalmente P. incarnata. No entanto, há pouco conhecimento acerca das espécies P. edulis e P. alata, sobretudo em relação à polpa e sementes. Os extratos da polpa e das sementes dos frutos dessas duas espécies de \"maracujá\", Passiflora alata e Passiflora edulis, foram estudados com o objetivo de identificar alcaloides harmânicos, pelo preparo das amostras por extração por sorção em barra magnética recoberta com polidimetilsiloxano (SBSE-PDMS) e SBSE recoberta com polietilenoglicol silicone (SBSE-EG Silicone) e análise por cromatografia líquida de alta eficiência com detector por fluorescência (CLAE-Flu) e cromatografia líquida de ultra eficiência acoplada à espectrometria de massas sequencial (CLUE-EM/EM). A análise dos alcaloides harmana e harmina nos extratos da polpa de P. alata foi feita por meio do método de adição de padrão e mostrou menor quantidade destes alcaloides, em comparação com os resultados da análise dos extratos da polpa dos frutos de P. edulis, no trabalho de Pereira e colaboradores. As análises CLUE-EM e CLUE-EM/EM possibilitaram a identificação dos alcaloides nos extratos: nas sementes de P. alata, os alcaloides harmana, harmina, harmol, harmalol e harmalina foram identificados, enquanto que na polpa, harmana e harmina tiveram a confirmação da sua presença. Nos extratos da polpa dos frutos de P. edulis observou-se os alcaloides harmana, harmina e harmalina. E nas sementes de P. edulis harmina foi encontrada, porém há indícios da presença de harmana. A literatura sobre as barras de SBSE-EG Silicone Twister&®; não relata nenhum estudo relacionado ao seu uso para extração e concentração de alcaloides harmânicos nos extratos de P. alata e P. edulis. Por isso foi proposto inicialmente a aplicação do planejamento fatorial fracionário para otimização do método de extração, utilizando os padrões comerciais dos alcaloides harmana e harmina. O planejamento experimental revelou as variáveis principais e seus níveis de importância, e a partir destes resultados foi realizado o estudo cinético dos tempos de extração e de dessorção das barras de SBSE-EG Silicone. Porém, os resultados mostraram que as barras de SBSE-EG Silicone não são adequadas para a extração dos alcaloides harmana e harmina, uma vez que a recuperação obtida foi baixa, na ordem de 30%. / \"Maracujá\" is the popular name given to the fruit of several species of Passiflora genus, from Passifloraceae family, it is widely commercialized and consumed around the world, and Brazil is one of the largest producers of this fruit. Some studies pointed out the possible toxicity related to Passiflora species, mainly P. incarnata. Although, there is a lack of knowledge about the P. edulis and P. alata species, especially with regards to the pulp and seeds. The extracts of pulp and seeds from the \"maracujá\" species Passiflora alata and Passiflora edulis, were studied in order to identify harman alkaloids. The samples were prepared by extraction with sorptive stir bar coated with polydimethylsiloxane (SBSE-PDMS) and SBSE coated with polyethylene glycol silicon (SBSE-EG Silicone). The samples were analyzed by high performance liquid chromatography with fluorescence detector (HPLC-Flu), and ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Harmane and harmine alkaloids in P. alata pulp extracts were analyzed using the standard addition method and the results showed a lower amount of these alkaloids, compared with the test results for the extracts from the P. edulis pulp in the work of Pereira et al. UHPLC-MS and UHPLC-MS/MS analysis enabled to identify the alkaloids amount present in the extracts. In the P. alata seeds extract the following alkaloids were identified harmane, harmine, harmol, harmalol and harmaline, while in the pulp extract, harmane and harmine were confirmed. In the extracts of P. edulis pulp the alkaloids identified were harmane, harmine and harmaline. And in the P. edulis seeds extract the harmine alkaloid was found, some indications of the presence of harmana were observed. The literature about SBSE-EG Silicone Twister® bars reports no study related to their use for extraction and concentration of harman alkaloids in P. alata and P. edulis. Thus, it was initially proposed the application of fractional factorial design to optimize the extraction method using commercial standards of harmane and harmine alkaloids. The experimental design revealed the main variables and their importance levels, and from these results kinetic studies were performed for the extraction and desorption times of SBSE-EG Silicone bars. However, the results showed SBSE-EG Silicone bars are not suitable for the extraction of harmane and harmine alkaloids, since the recovery obtained was low, on the order of 30%.

In food security area, the application of good agricultural practices is a growing concern for public health in the Brazilian domestic market and for the competitiveness countries in the external market. To ensure safety of food from animal origin, monitoring is required and Maximum Residue Limits (MRLs) must be evaluated. Therefore, the development of appropriate analytical methods for residues determination is necessary. In this work a simple, fast and efficient multiclass method of sample preparation was developed for the determination of veterinary drugs residues in bovine kidney, liver and muscle. Determination step was performed by ultra-high-performance liquid chromatographic–tandem mass spectrometry (UHPLC-MS/MS). UHPLC-MS/MS and sample preparation conditions were optimized using experimental designs. Extraction and clean up were performed by solid-liquid extraction and dispersive solid-phase extraction (d-SPE). Central composite designs were used in order to optimize the clean up step. The proposed method was validated using acetonitrile as solvent extraction followed by clean up with EMR-Lipid® sorbent and aqueous solution of 5% trichloroacetic acid (m/v). The proposed method was validated according to the criteria of the European Commission Decision 2002/657/EC. Linearity presented r2 ≥ 0.99 for most the evaluated compounds and recoveries values and RSD in the range recommended by EU. Decision limit (CCα) and detection capability (CCβ) presented values around the maximum residue limits (MRL) of each compound. Monensin was the only compound that did not present satisfactory results for bovine kidney and muscle. The developed sample preparation followed by UHPLC-MS/MS analysis was efficient for the determination of veterinary drug residues in bovine liver, kidney and muscle. The proposed methodology has been successfully applied in real samples and also in proficiency test and proved to be a great option for routine analysis. / No âmbito da segurança dos alimentos a aplicação das boas práticas agropecuárias é uma preocupação crescente tanto para a saúde pública no mercado interno brasileiro quanto para à competitividade do país no mercado externo. Para garantir a inocuidade dos alimentos de origem animal são realizados monitoramentos em diferentes tipos de amostras e adotados Limites Máximos de Resíduo (LMR). Diante disso, se faz necessário o desenvolvimento de métodos analíticos adequados para determinação de resíduos. Neste trabalho desenvolveu-se um método multiclasse de preparo de amostras simples, rápido e eficaz para a determinação de resíduos de medicamentos veterinários em rim, fígado e músculo bovino. A etapa de determinação foi realizada por cromatografia líquida de ultra eficiência acoplada à espectrometria de massas em série (UHPLC-MS/MS). O sistema UHPLC-MS/MS e a etapa de preparo de amostra foram otimizados com auxílio de planejamento experimental. As etapas de extração e limpeza do extrato foram realizadas por extração sólido-líquido e extração em fase sólida dispersiva (d-SPE). Planejamentos composto central foram utilizados para otimização da etapa de limpeza do extrato. O procedimento otimizado consistiu de extração por acetonitrila, seguido de limpeza com o sorvente EMR-Lipid® e solução aquosa de 5% (m/v) ácido tricloroacético. O método proposto foi validado de acordo com os critérios de referência da Decisão 2002/657/CE da Comunidade Europeia. A linearidade apresentou r2 ≥ 0,99 para maioria dos compostos avaliados e os valores de recuperação e RSD estão na faixa recomendada pela Comunidade Europeia. O limite de decisão (CCα) e a capacidade de detecção (CCβ) apresentaram valores em torno dos LMR de cada composto. Apenas a monensina não obteve resultados satisfatórios para rim e músculo bovino. O preparo de amostra desenvolvido seguida de análise por UHPLC-MS/MS foi eficiente para a determinação de resíduos de medicamentos veterinários em rim, fígado e músculo bovino. A metodologia proposta foi aplicada com sucesso em amostras reais e também em ensaio de proficiência e provou ser uma ótima opção para análise de rotina.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2018-05-17T18:37:10Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-05-22T11:40:51Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2018-05-22T11:40:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-07-27 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A artepilina C é o principal marcador químico utilizado no controle de qualidade da própolis verde, a qual é produzida especialmente em Minas Gerais e comercializada principalmente para o Japão e que possui como principal fonte botânica a planta Baccharis dracunculifolia DC. (Asteraceae). Entre as técnicas que podem ser utilizadas para a análise da artepilina C, a cromatografia líquida de ultra eficiência (CLUE) acoplada a espectrometria de massas (EM) apresenta inúmeras vantagens. Em relação às suas atividades biológicas, a artepilina C apresenta ação antioxidante e antitumoral, não tendo sido ainda avaliada sua embriotoxicidade e atividade antienvelhecimento. O presente trabalho teve como objetivos isolar a artepilina C, desenvolver e validar método analítico de quantificação dessa substância por CLUE-EM-Q-TOF, bem como determinar sua atividade antienvelhecimento em nematoides Caenorhabditis elegans e sua toxicidade em embriões de Zebrafish. Para isolar a artepilina C de amostra de própolis verde, foi realizada extração básica seguida de acidificação, com posterior fracionamento por cromatografia líquida a vácuo (CLV) e cromatografia líquida de alta eficiência (CLAE) semipreparativa. Para identificação da substância, foi realizada análise por CLAE e ressonância magnética nuclear (RMN) de 1H e 13C. Após isolamento, a substância foi utilizada como padrão para validação de metodologia de quantificação da artepilina C em própolis verde por CLUE-EM-Q-TOF. A metodologia mostrou-se específica, linear, precisa, exata e robusta nas condições avaliadas. Em relação aos ensaios biológicos frente ao nematoide C. elegans, a artepilina C não foi tóxica ao animal, em nenhuma das concentrações avaliadas. Contudo, a artepilina C (125 μM) não foi capaz de prolongar seu tempo de vida. Em embriões de Zebrafish, a substância foi tóxica nas concentrações de 250, 125 e 50 μM, indicando a necessidade de estudos mais aprofundados quanto à sua embriotoxicidade. / Artepillin C is the main chemical marker used in quality control of green propolis, which is produced especially in Minas Gerais and marketed mainly to Japan, being Baccharis dracunculifolia DC. (Asteraceae) its main botanical source. Among the techniques that can be used for the analysis of artepillin C, ultra high performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS) has many advantages. Regarding its biological activities, artepillin C shows antioxidant and antitumor action, and its embryotoxicity and antiaging activity have not been evaluated yet. The aim of this work was to isolate artepillin C, to develop and validate the analytical method of quantification of this substance by UHPLC-MS-Q-TOF, as well as to determine its antiaging activity in Caenorhabditis elegans nematodes and its toxicity in Zebrafish embryos. To isolate the artepillin C from green propolis sample, basic extraction was performed followed by acidification, with subsequent fractionation by vacuum liquid chromatography (VLC) and semipreparative high performance liquid chromatography (HPLC). HPLC and nuclear magnetic resonance (NMR) 1H and 13C analysis were performed for the substance identification. After isolation, the substance was used as standard for validation of quantification methodology of artepillin C in green propolis by UHPLC-MS-Q-TOF. The methodology was specific, precise, accurate and robust for the evaluated conditions. About the biological assays in C. elegans nematode, artepillin C was not toxic for the animal at any of evaluated concentrations. However, artepillin C (125 μM) was not able to prolong its life span. In Zebrafish embryos, the substance was toxic at 250, 125 e 50 μM concentrations, showing the need for further studies about its embryotoxicity.

The aim of this diploma thesis was the development of a screening method for analysis of 17 benzodiazepines in urine samples using ultra high-performance liquid chromatography with tandem mass spectrometric detection. The partial task was to optimize the conditions for the enzymatic hydrolysis of benzodiazepine glucuronides present in urine using design of experiments (DOE). The optimized chromatographic system consisted of a Zorbax Eclipse Plus Phenyl-Hexyl RRHD column (100 × 2.1 mm, 1.8 μm) and mobile phase consisting of water with 0.1 % acetic acid (component A) and acetonitrile with 0.1 % acetic acid (component B) in various ratios according to the gradient program. Flow rate was 0.2 ml/min, column temperature was 40 řC, and total analysis time was 12 min. Calibration curves for all analytes were measured under optimized conditions in methanol and urine. After optimal detection conditions for oxazepam-glucuronide were found, oxazepam glucuronide was hydrolysed using β-glucuronidase from the abalone to confirm the functionality of the enzyme within the pilot experiment. Optimization of enzymatic hydrolysis conditions via 27 experiments proposed by program Minitab 16 using the Box-Behnken design will be realized later.