Dissertations / Theses on the topic 'Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC-MS/MS)'

Orientador: Isabel Cristina Sales Fontes Jardim
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química
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Resumo: Este trabalho envolve o desenvolvimento, a otimização e a validação de um método analítico para determinação de multirresíduos de agrotóxicos em amostras de morango, por cromatografia líquida acoplada à espectrometria de massas sequencial (LC-MS/MS). No preparo de amostra utilizou o método QuEChERS (quick, easy, cheap, effective, rugged and safe), que foi testado nas três versões, Original, AOAC Official Method e European Committee for Standardization (CEN) Standard Method EN 15662, além da versão CEN 15662 modificada. Também foram otimizados os solventes de extração, massas do agente secante e, na etapa de clean-up por extração em fase sólida dispersiva (d-SPE), o sorvente comercial PSA (primary secondary amine), alguns preparados no laboratório à base de polímeros de siloxano, como octadecil, octil, amino, fenil, e a mistura PSA e octadecil. As avaliações dos métodos foram baseadas, principalmente, nos valores de recuperação e nos estudos sobre o uso de diferentes sorventes, outros parâmetros que estimam a eficiência do clean-up também foram utilizados, como aspecto físico do extrato final, quantidade de coextratos da matriz, obtida por medidas gravimétricas, e efeito matriz. O método desenvolvido foi validado por meio dos parâmetros analíticos de seletividade, limite de detecção (LOD), limite de quantificação (LOQ), linearidade, exatidão e precisão, conforme o guia Sanco para análises de resíduos de agrotóxicos em alimentos e, posteriormente, amostras comerciais de morango da região de Campinas foram analisadas. O método validado por LC-MS/MS apresentou-se seletivo, preciso, exato e atingiu concentrações abaixo dos respectivos limites máximos de resíduos (LMR) para determinação de agrotóxicos em morango. Este método foi transferido para cromatografia líquida de ultra eficiência (UHPLC), que mostrou redução no tempo de análise, na vazão da fase móvel (FM) e no volume de injeção de amostra e da FM, e similaridade na detectabilidade dos analitos
Abstract: This work involves the development, optimization and validation of an analytical method for multiresidue determination of pesticides in strawberry samples by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Sample preparation used the QuEChERS (quick, easy, cheap, effective, rugged and safe) method, which was tested in three versions, Original, AOAC Official Method and European Committee for Standardization (CEN) Standard Method EN 15662, and also CEN 15662 modified version. The factores optimized were extraction solvents, amount of drying agent and in the clean-up step by dispersive solid phase extraction (d-SPE), the commercial sorbent PSA (primary secondary amine), several prepared in the laboratory based on siloxane polymers, such as octadecyl, octyl, amine, phenyl, and the mixture PSA and octadecyl. The evaluation of the methods was based mainly on the recovery values and for the study of different sorbents, other parameters that estimate the efficiency of the clean-up were also used such as the physical aspect of the final extract, the amount of interference matrix obtained using gravimetric measurements, and the matrix effect. The developed method was validated by the analytical parameters of selectivity, limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy and precision, as described in the Sanco guide for analysis of pesticide residues in foods. After, commercial strawberry samples from the Campinas region were analyzed. The validated method by LC-MS/MS was selective, precise, accurate and reached levels below the respective maximum residue limits (MRLs) for the determination of pesticides in strawberries. This method was transferred to ultra high performance liquid chromatography (UHPLC), which showed a reduction in analysis time, the mobile phase (MP) flow rate and the injection volume of the sample and MP, and similarity in the detectability of the analytes
Doutorado
Quimica Analitica
Doutora em Ciências

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
A rapid, specific and highly sensitive liquid chromatography-tandem mass spectrometry method was developed to determine nimodipine in human plasma using dibucaine as the internal standard (IS) is described. The analyte (m/z 418,6 > 342,6) and IS (m/z 344,2 > 271,0) were extracted from plasma samples by liquid-liquid extraction using hexane-ethyl acetate (1:1v/v). Chromatography was performed on a Varian Polaris C18 analytical column (3 micrometer, 50 x 2,0 mm) and pre-column SecurityguardTM C18 (4,0 x 3,0 mm). The phase mobile consisted of Acetonitrile-Ammonium acetate 0.02 ml/L (80:20v/v). The method had a chromatographic run time of 4.5 min and linear calibration curve over the range 0.1- 40 ng/mL (r2 > 0.9938). The limit of quantification (LQ) was 0.1 ng/mL. The intra-day precision for the limit quantification was 0.00% (batch 01), 5.71% (batch 02) and 5.27% (batch 03); for the quality controls low (QCL), middle (QCM) and high (QCH) the results were respectively 8.57, 0.81 and 1.37%. The inter-day precision for LQ and QCL, QCM and QCH were respectively: 7% and 5.46, 4.12 and 3.37%. The intra-day accuracy for LQ was 110, 96 and 104%; for QCL, QCM and QCH the results were100.67, 109.09 and 109.72% respectively. The results of the inter-day accuracy for LQ, QCL, QCM and QCH were respectively and 110.0, 96.0, 104.0% for the limit of quantification and 8.57, 0.81, 1.37% and 100.67, 109.09, 109.72% respectively: 103% e 102.89, 106.60, 109.69%. This validated method was successfully applied for the pharmacokinetic profiles of nimodipine tablets administered to 24 healthy volunteerâs participant of bioavailability comparative study. Geometric mean of Test formulation/Refernce formulation individual percent ratio was 104,56% for AUC0-48h and 55,73% for Cmax. The 90% for the confidence intervals (CI) were 94,80-115,32% e 44,73-69,42%, respectively. The values of half-life and Cmax for test formulation and reference formulation were 27,83;32,78h and 9,48;18,76ng/mL, respectively. The values of Tmax were 2,34;0,98h for the formulations test and reference respectively. Since the 90% CI for Cmax and AUC0-48h, were within the 80-125% interval proposed by the âFood and Drug Administrationâ and ANVISA, it was concluded that the two formulations of nimodipine 30mg tablets were not bioequivalent, according to the rate of absorption after single dose administration.
Um mÃtodo rÃpido, sensÃvel e especÃfico de Cromatografia LÃquida de Alta EficiÃncia acoplada à Espectrometria de Massa (LC-MS-MS) foi desenvolvido para determinar nimodipino (analito) em plasma humano usando dibucaÃna como padrÃo interno (PI). O analito (m/z 418,6 > 342,6) e o PI (m/z 344,2 > 271,0) foram extraÃdos de amostras de plasma atravÃs de extraÃÃo lÃquido-lÃquido utilizando hexano-acetato de etila (1:1v/v). As corridas cromatogrÃficas foram executadas utilizando-se uma coluna analÃtica Varian Polaris C18 (3 micrÃmetros, 50 x 2,0 mm) e uma prÃ-coluna SecurityguardTM C18 (4,0 x 3,0 mm). A fase mÃvel consistiu de acetonitrila-soluÃÃo de acetato de amÃnio 0,02 mol/L (80:20v/v). O mÃtodo teve um tempo total de corrida de 4,5 min e uma curva de calibraÃÃo linear que variou de 0,1-40 ng/mL. O limite de quantificaÃÃo de 0,1 ng/mL. A precisÃo intra-ensaio para o limite de quantificaÃÃo (LQ) foi 0,00% (lote 01), 5,71% (lote 02) e 5,27% (lote 03); para os controles de qualidade baixo (CQB), mÃdio (CQM) e alto (CQA) os resultados foram respectivamente: 8,57, 0,81 e 1,37%. A precisÃo interensaio para o LQ e os CQB, CQM e CQA foram respectivamente de: 7% e 5,46, 4,12 e 3,37%. A exatidÃo intra-ensaio para o LQ foi 110, 96 e 104%; para CQB, CQM e CQA os resultados foram 100,67, 109,09 e 109,72% respectivamente. Os resultados da exatidÃo interensaio para o LQ, CQB, CQM e CQA foram respectivamente de: 103% e 102,89, 106,6, 109,69%. Este mÃtodo foi aplicado para a avaliaÃÃo do perfil farmacocinÃtico do nimodipino administrado em 24 voluntÃrios sadios participantes de um estudo de biodisponibilidade comparativa. A mÃdia geomÃtrica da FormulaÃÃo teste/FormulaÃÃo referÃncia para as porcentagens individuais foi 104,56% para ASC0-48h e 55,73% para Cmax. Os intervalos obtidos a partir do intervalo de confianÃa (IC) de 90% foram 94,80-115,32% e 44,73-69,42% respectivamente. Os valores de meia-vida e Cmax para as formulaÃÃes teste e referÃncia foram de 27,83;32,78h e 9,48;18,76ng/mL, respectivamente. Os valores de Tmax foram de 2,34;0,98h para as formulaÃÃes teste e referÃncia, respectivamente. Considerando o IC de 90% para Cmax e ASC0-48h dentro da variaÃÃo de 80-125% proposto pelo Food and Drug Administration e ANVISA, as duas formulaÃÃes de nimodipino 30mg nÃo sÃo bioequivalentes quanto à taxa de absorÃÃo (Cmax) apÃs uma Ãnica administraÃÃo.

Per- and polyfluorinated alkyl substances (PFASs) belong to a large group of man-made chemicals that pollute the environment. Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) are the most commonly found PFASs. The pollution of PFASs can be caused among others by using of aqueous fire-fighting foams (AFFFs). PFASs are persistent compounds; that can travel long distances and bioaccumulate in biota. There are several exposure routes for PFASs, but the most common are via food and drinking water. A possible way for PFASs to enter the food chain is by adsorption to microbes. In this project, binding of PFASs to three gram-negative bacteria, Eschericha coli, Acidovorax delafieldii and Pseudomonas nitroreducens, was assessed. Microbes were exposed for fluorinated compounds in environmental water samples and a PFAS-11 solution with 11 PFAS substances prepared in the laboratory. The binding seems to be preferential to the most abundant compounds, PFOS, since the second most abundant compound in the samples was PFHxS with concentrations at one third of the PFOS concentration but nonetheless PFHxS was not detected in any of the samples. The binding of mainly one PFAS was identified; PFOS was bound at highest concentrations in E. coli treated with both environmental water sample and a PFAS-11 solution. Low concentrations of FOSA and PFDoDS were identified in E. coli and PFNA in A. delafieldii. Only PFOS was detected in P. nitroreducens. The concentrations of other PFASs were below their respective method detection limits.

A venlafaxina (VEN), em razão de sua eficácia e brandos efeitos adversos, tem sido um dos antidepressivos mais prescritos no tratamento da depressão e ansiedade. Neste trabalho, um método analítico empregando as técnicas MISPE miniaturizada e cromatografia líquida acoplada à espectrometria de massas em Tandem, foi utilizado para a determinação de VEN e seus principais metabólitos em amostras de plasma para fins de monitorização terapêutica. A fase MIP foi sintetizada via polimerização radicalar por precipitação, fazendo uso de VEN (molécula molde), ácido metacrílico (monômero funcional), etileno glicol dimetacrilato, (reagente reticulante) e 2,2 azobisisobutironitrila (iniciador radicalar) em tolueno (solvente). Para controle utilizou-se o polímero não impresso (NIP), sintetizado por procedimento análogo ao do MIP, porém sem o uso da molécula molde. A caracterização química e estrutural dos polímeros foi realizada por espectroscopia no infravermelho com transformada de fourier e microscopia eletrônica de varredura. A otimização das variáveis de MISPE miniaturizada favoreceu a detectabilidade analítica e diminuiu o efeito de memória. As extrações realizadas com MIP apresentaram taxa de recuperação de 84% para VEN e de 2-28% para os antidepressivos (clorpromazina, fluoxetina, clomipramina, imipramina e sertralina). O polímero não impresso apresentou baixa recuperação para a VEN (taxa de recuperação: 49%) e para os demais antidepressivos (taxas de recuperação menores que 40%). Estes experimentos comprovam a seletividade da fase MIP desenvolvida. O método padronizado apresentou linearidade na faixa de 3 a 700 ng mL-1 para VEN, 5 a 700 ng mL-1 para O-desmetilvenlafaxina (ODV) e de 3 a 500 ng mL-1 para N-desmetilvenlafaxina (NDV), precisão com coeficientes de variação menores que 15% e exatidão com valores de erro padrão relativo na faixa de -11,8 a 16,01 %. As concentrações correspondentes aos limites inferiores de quantificação para VEN (3 ng mL-1) e ODV ( 5 ng mL-1) foram inferiores aos intervalos terapêuticos preconizados. O método desenvolvido, quando comparado a aos métodos da literatura para determinação de VEN e metabolitos, apresentou maior seletividade, menor consumo de amostra e de solventes orgânicos e permitiu a reutilização da fase extratora. Segundo os parâmetros de validação analítica avaliados e amostras de pacientes em terapia com VEN analisadas, o método proposto é adequado para determinação de VEN, ODV e NDV em amostras de plasma para fins de monitorização terapêutica.
Venlafaxine elicits a small number of adverse effects, so it is one of the most frequently prescribed drugs to treat major depression, generalized anxiety, and social anxiety disorders in adults. In this study, venlafaxine (VEN), O-desmethylvenlafaxine (ODV), and N-desmethylvenlafaxine (NDV) were pre-concentrated with the aid of miniaturized SPE based on MIPs as extraction phase. MIPs are synthetic polymers with cavities specifically designed to hold a target molecule or structurally similar compounds. The molecularly imprinted polymers were prepared by addition of VEN, metacrylic acid (MAA, monomer), ethylene glycol dimethacrylate (EGDMA, cross-linker), and 2,2-azobisisobutyronitrile (AIBN, initiator) to toluene (solvent). The non-imprinted polymer (NIP), used for comparison, was also synthesized by following exactly the same procedure, but excluding the template VEN from the formulation. The polymer was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy (SEM). Optimization of the MIP phase extraction variables favored miniaturized analytical detectability and reduced the memory effect. The extractions performed with the synthesized MIP showed recovery rate of 84% for VEN and 2-28% for other antidepressants (chlorpromazine, fluoxetine, clomipramine, imipramine, and sertraline). The non-imprinted polymer provided low recovery of VEN (recovery rate: 49%) and other antidepressants (recovery rates lower than 40%). These experiments demonstrated the selectivity of the developed MIP phase. The standardized method was linear in the range of 300 - 700 ng mL-1 for VEN, 5-700 ng mL-1 for ODV, and 3 to 500 ng mL-1 for NDV. Precision had coefficients of variation smaller than 15%; the accuracy standard error values ranged from -11.8 to 16.01%. Compared with literature methods, the developed method was more selective for determination of VEN and metabolites, required lower consumption of sample and organic solvents, and enabled reuse of the extraction phase. According to the assessed analytical validation parameters and to the analysis of samples obtained from patients undergoing therapy with VEN, the proposed method is suitable to determine VEN, NDV, and ODV in plasma samples for therapeutic drug monitoring.

En raison de la diffusion incontrôlée sur le e-commerce, la sécurité et l’alternative légale aux stupéfiants habituels, les nouvelles substances psychoactives (NPS), d’apparition récente (2008), sont au cœur des phénomènes récents d’addiction et de décès mal expliqués. Au-delà des différents défis dans nos sociétés (prévention, législation), la capacité d’identifier les NPS dans des échantillons biologiques pour caractériser leur utilisation, présente de nombreux challenges analytiques. L’objectif principal de cette thèse a été de collecter des échantillons biologiques (sang, urine, cheveux) provenant de cas d’exposition à des NPS et d’y caractériser les substances présentes à l’aide de méthodes analytiques originales, dans le but d’enrichir les librairies de spectres de masse et d’améliorer, en conséquence, la détection de la consommation de NPS. En particulier, il s’agissait d’augmenter la fenêtre de détection de la prise de NPS en se focalisant sur les métabolites qui sont, le plus souvent, les produits majeurs d’élimination. Le développement analytique, par chromatographie liquide ultra haute performance couplée à la spectrométrie de masse en tandem (UHPLC-MS/MS), a demandé plusieurs mois d’optimisation afin d’obtenir une méthode robuste, exhaustive et sensible. Actuellement, la librairie de spectres MS comporte 114 NPS et est mise à jour régulièrement. A la suite de ce développement, ma thèse a porté sur l’étude de cas d’intoxication vus au service des urgences du CHU de Strasbourg, mais aussi en médecine légale, avec des situations de décès et d’identification de produits inconnus provenant de saisies (poudres et cristaux). Il a également été nécessaire de développer des outils analytiques complémentaires, tels que la caractérisation de métabolite(s) par étude sur microsomes hépatiques humains (HLMs), et l’utilisation de la spectroscopie par résonance magnétique nucléaire (RMN) afin d’identifier avec certitude certains composés et de déterminer leur degré de pureté. Les outils analytiques développés et la stratégie mise en place ont permis la rédaction de 18 publications, ainsi que l’agencement de nombreuses collaborations
Due to the uncontrolled spread on the Internet and their legal alternative to usual drugs, the new psychoactive substances (NPS), recently appeared (2008), are at the center of recent phenomena of addiction and badly explained deaths. Beyond different challenges in our societies (prevention, legislation), the ability to identify NPS in biological samples, in order to characterize their use, presents many analytical challenges. The main objective of this thesis was to collect biological samples (blood, urine, hair) from cases of exposure to NPS and to characterize the substances present using original analytical methods, in order to enlarge the libraries of mass spectra and improve, as a result, the detection of NPS consumption. In particular, it was intended to increase the detection sensitivity of NPS intake by focusing on the metabolites that are often the major products of elimination. This analytical development, by ultra-high liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), required several months of optimization in order to obtain a robust, exhaustive and sensitive method. At present, the mass spectra database has 114 NPS and is regularly updated. Thereafter, ma thesis focused on the study of cases of intoxication observed in the emergency department of Strasbourg, but also in legal medicine with situations of deaths and identification of unknown products collected from seizures (powders and crystals). It has also been necessary to implement complementary analytical tools, such as the characterization of metabolites by human liver microsomes (HLMs), and the use of nuclear magnetic resonance (NMR) spectroscopy to accurately identify the compounds and establish their purity degrees. The analytical tools developed, and the strategy adopted, allowed the writing of 18 publications, as well as the setting up of numerous collaborations

>Magister Scientiae - MSc
Subsistence farmers may contribute significantly to food production, food security, and employment in South Africa. However poor storage practices and contamination with mycotoxins, particularly fumonisins and aflatoxins impacts adversely on production, food safety and food security. Mycotoxins are toxic natural food-borne compounds which frequently contaminate agricultural produce worldwide. They are hazardous to humans and animals and result in significant production losses for farmers. This study focused on former Bantustans in Northern South Africa, namely Vhembe District Municipality (Limpopo) and Gert Sibande District Municipality (Mpumalanga). The aim was to assess mycological and mycotoxin contamination of crops grown by subsistence farmers. A semi-structured questionnaire was administered to randomly thirty-nine households. Data on demographics, storage practices and production during period of 2011 and 2012 cropping seasons were collected. One hundred and fifteen (115) crop samples (maize, beans and peanuts) were collected for analysis. Standard mycological methods and validated mycotoxin analysis methods (HPLC and LC- MS/MS) were used. It was found that maize was the staple food in both provinces, with a significant difference (p = 0.0184) in its production between the two districts; Vhembe produced 0.6 tonnes compared to 2.4 tonnes in Gert Sibande. The majority of the farmers for storage used traditional open wooden cribs (15/20) and steel tanks (5/20) while VDM farmers used sealed store houses 5/19 and 15/19 used polystyrene sacks. Aflatoxin occurrence was low with <1% of GSDM samples contaminated compared to 11% of VDM samples. No significant difference (p > 0.05) was observed in the aflatoxin contamination in VDM samples between the year 2011 and 2012. Samples from VDM households had higher Aspergillus fungal infection (maximum incidence 69%) compared to GSDM (27%) over both seasons. The most frequently isolated Fusarium species in VDM samples was F. verticillioides (92%; 93%), and F. subglutinans (97%; 80%) in GSDM samples over seasons 2011 and 2012, respectively. Highest levels of fumonisins (FB1+ FB2) ranged between 1010 μg/kg and 12168 μg/kg with less than 30% extremely contaminated above the regulated limit in 91% of samples from Limpopo over both seasons (2011 and 2012). Fumonisin levels between the two seasons in VDM showed no significant difference (p>0.05). Only three (less than 5%) from 68% GSDM contaminated maize samples were above the FB1 and FB2 limit. In 2011, there were two highly contaminated maize samples (1762 μg/kg and 4598 μg/kg) with the other samples less than 600 μg/kg, whereas in season two (2012) all samples were below 200 μg/kg, except one highly contaminated sample (26115 μg/kg). None of the beans and peanuts from Mpumalanga was contaminated with mycotoxins above the recommended limit, but from Limpopo 1/5 peanuts was found contaminated with aflatoxin G1 (41 μg/kg). Natural occurrence and contamination of both fumonisin and aflatoxin in stored home-grown maize from VDM was significantly (p < 0.0001) higher than GSDM over both seasons. In general, Limpopo farmers’ experience lower harvests and greater mycotoxin contamination of agricultural produce. This may be attributed in part to poor storage practices and environmental and climatic conditions in that agro-ecological zone.

Orientador: Alexandra Christine Helena Frankland Sawaya
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A palavra própolis tem origem Grega: pro significa em defensa de, e polis significa comunidade. A própolis é usada pelas abelhas para fortalecer as paredes da colmeia e para cobrir as paredes internas e por devido a sua atividade antimicrobiana. A composição química da própolis é variável segundo a biodiversidade e a origem geográfica. A própolis vermelha, encontrada no nordeste e norte do Brasil, apresenta promissoras atividades biológicas: atividade antimicrobiana, antiparasitária, antioxidante, citotóxica, antiinflamatória, analgésica, efeitos antiobesidade, contra psoríase e hepatoprotetores. A composição química da própolis vermelha, relatada na literatura, parece variar qualitativa e quantitativamente, inclusive entre amostras coletadas na mesma região. Isto pode ser devido a variações sazonais, a flora direitamente ao redor das colméias ou aos métodos de análise. A maioria das classes de substâncias já identificadas pode ser analisada adequadamente por espectrometria de massas com ionização por eletrospray e cromatografia líquida. Os constituintes de média e alta polaridade (como fenólicos, flavonóides e benzofenonas) são responsáveis por muitas das atividades biológicas reportadas à própolis vermelha. A cromatografia líquida de ultra-eficiência acoplada à espectrometria de massas (UHPLC-MS) é um método moderno, rápido e sensível. Permite avaliar os perfis químicos das amostras e determinar sua composição qualitativamente, comparando os espectros de massas (MS/MS) dos componentes das amostras com informações na literatura. Portanto UHPLC-MS é a ferramenta mais adequada para a avaliação da composição de matrizes complexas como a própolis. A comparação do perfil químico de diversas amostras de própolis vermelha brasileira e a avaliação de sua atividade antimicrobiana e antioxidante permitirá identificar substâncias que possam estar contribuindo para sua bioatividade, e levar á identificação de marcadores químicos para o controle de qualidade destas amostras, bem como possibilitará definir se há um ou vários tipos de própolis vermelha
Abstract: The word, propolis is of Greek origin: pro means in defense of and polis means community. Propolis is used by bees to strengthen the hive walls and cover the inner walls for its antimicrobial activity. The chemical composition of propolis varies depending on the biodiversity and geographic origin. Red propolis from the north and northeast of Brazil shows promising biological activity: antimicrobial, antiparasitic, antioxidant, cytotoxic, anti-inflammatory, analgesic, antiobesity, antipsoriasic and hepatoprotective effects. The chemical composition of red propolis reported in literature seems to vary qualitatively and quantitatively, even between samples collected in the same region. This may be due to seasonal variations, the flora directly around the hives or differences in the analytical methods. Most of the compounds already identified can be analyzed by mass spectrometry with electrospray ionization and liquid chromatography. These compounds of medium and high polarity (phenolics, flavonoids and benzophenones) are responsible for most of the biological activities reported. Ultra efficient liquid chromatography coupled with mass spectrometry (UHPLC-MS) is a modern, fast and sensitive method. It allows the characterization of the chemical profile of the samples and determines their qualitative composition, by comparison of the mass spectra (MS/MS) of the compounds with data from literature. Therefore it is the most adequate tool to evaluate the composition of complex matrixes such as propolis. The comparison of the chemical profile of diverse red propolis samples and evaluation of their antimicrobial and antioxidant activities allow us to identify the compounds responsible for these activities, indicate the marker compounds and define if there is one type, or several types, of red propolis
Mestrado
Fármacos, Medicamentos e Insumos para Saúde
Mestra em Ciências

This thesis deals with the content of adaptogens in vegetable Schizandra chinensis. The theoretical part deals with the plant Schisandra chinensis, its occurrence, characteristics, uses and cultivation. Location of plants Schisandra chinensis are classified according to Köppen climate classification and compared with the climate in the Czech Republic, due to possible prediction of growing plants in the country. Further are described adaptogens (bioactive substances) contained in this plant and to methods for their extraction and analysis. The experimental part of the thesis deals with the identification of schisandrin in the plant Schisandra chinensis grown in our conditions. The assessment was performed by ultra-performance liquid chromatography (UPLC), high-performance liquid chromatography (HPLC) and direct injection into tha mass spectrometry (MS). Schisandrin was successfully identified in all the samples.

Doutoramento em Química
This thesis reports the application of metabolomics to human tissues and biofluids (blood plasma and urine) to unveil the metabolic signature of primary lung cancer. In Chapter 1, a brief introduction on lung cancer epidemiology and pathogenesis, together with a review of the main metabolic dysregulations known to be associated with cancer, is presented. The metabolomics approach is also described, addressing the analytical and statistical methods employed, as well as the current state of the art on its application to clinical lung cancer studies. Chapter 2 provides the experimental details of this work, in regard to the subjects enrolled, sample collection and analysis, and data processing. In Chapter 3, the metabolic characterization of intact lung tissues (from 56 patients) by proton High Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy is described. After careful assessment of acquisition conditions and thorough spectral assignment (over 50 metabolites identified), the metabolic profiles of tumour and adjacent control tissues were compared through multivariate analysis. The two tissue classes could be discriminated with 97% accuracy, with 13 metabolites significantly accounting for this discrimination: glucose and acetate (depleted in tumours), together with lactate, alanine, glutamate, GSH, taurine, creatine, phosphocholine, glycerophosphocholine, phosphoethanolamine, uracil nucleotides and peptides (increased in tumours). Some of these variations corroborated typical features of cancer metabolism (e.g., upregulated glycolysis and glutaminolysis), while others suggested less known pathways (e.g., antioxidant protection, protein degradation) to play important roles. Another major and novel finding described in this chapter was the dependence of this metabolic signature on tumour histological subtype. While main alterations in adenocarcinomas (AdC) related to phospholipid and protein metabolisms, squamous cell carcinomas (SqCC) were found to have stronger glycolytic and glutaminolytic profiles, making it possible to build a valid classification model to discriminate these two subtypes. Chapter 4 reports the NMR metabolomic study of blood plasma from over 100 patients and near 100 healthy controls, the multivariate model built having afforded a classification rate of 87%. The two groups were found to differ significantly in the levels of lactate, pyruvate, acetoacetate, LDL+VLDL lipoproteins and glycoproteins (increased in patients), together with glutamine, histidine, valine, methanol, HDL lipoproteins and two unassigned compounds (decreased in patients). Interestingly, these variations were detected from initial disease stages and the magnitude of some of them depended on the histological type, although not allowing AdC vs. SqCC discrimination. Moreover, it is shown in this chapter that age mismatch between control and cancer groups could not be ruled out as a possible confounding factor, and exploratory external validation afforded a classification rate of 85%. The NMR profiling of urine from lung cancer patients and healthy controls is presented in Chapter 5. Compared to plasma, the classification model built with urinary profiles resulted in a superior classification rate (97%). After careful assessment of possible bias from gender, age and smoking habits, a set of 19 metabolites was proposed to be cancer-related (out of which 3 were unknowns and 6 were partially identified as N-acetylated metabolites). As for plasma, these variations were detected regardless of disease stage and showed some dependency on histological subtype, the AdC vs. SqCC model built showing modest predictive power. In addition, preliminary external validation of the urine-based classification model afforded 100% sensitivity and 90% specificity, which are exciting results in terms of potential for future clinical application. Chapter 6 describes the analysis of urine from a subset of patients by a different profiling technique, namely, Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Although the identification of discriminant metabolites was very limited, multivariate models showed high classification rate and predictive power, thus reinforcing the value of urine in the context of lung cancer diagnosis. Finally, the main conclusions of this thesis are presented in Chapter 7, highlighting the potential of integrated metabolomics of tissues and biofluids to improve current understanding of lung cancer altered metabolism and to reveal new marker profiles with diagnostic value.
A presente tese reporta a aplicação da metabolómica ao estudo de tecidos e biofluidos humanos (plasma sanguíneo e urina), com o intuito de caracterizar a assinatura metabólica do cancro pulmonar primário. No Capítulo 1, apresenta-se uma breve introdução sobre a epidemiologia e a patogénese deste tipo de cancro, bem como um sumário das principais alterações metabólicas tipicamente associadas ao cancro em geral. Descreve-se ainda a abordagem metabolómica, nomeadamente os métodos analíticos e estatísticos utilizados, assim como o estado da arte da sua aplicação em estudos clínicos do cancro do pulmão. No Capítulo 2, apresentam-se os detalhes experimentais deste trabalho, no que diz respeito ao grupo de indivíduos envolvidos, à colheita e análise das amostras e ao posterior tratamento dos dados. O Capítulo 3 descreve a caracterização metabólica de tecidos do pulmão (de 56 doentes) por espetroscopia de Ressonância Magnética Nuclear (RMN) de alta resolução com rotação no ângulo mágico. Após a otimização cuidada das condições de aquisição e a identificação detalhada dos sinais espetrais (mais de 50 metabolitos identificados), os perfis metabólicos dos tumores e dos tecidos adjacentes não envolvidos (controlos) foram comparados por análise multivariada, tendo sido discriminados com uma exatidão de 97%. Os metabolitos que mais significativamente contribuíram para esta diferenciação foram: glucose e acetato (diminuídos nos tumores), lactato, alanina, glutamato, GSH, taurina, creatina, fosfocolina, glicerofosfocolina, fosfoetanolamina, nucleótidos de uracilo e péptidos (aumentados nos tumores). Algumas destas variações corroboraram alterações típicas do metabolismo do cancro (e.g., glicólise e glutaminólise aumentadas), enquanto outras sugeriram novas pistas sobre a possível relevância de processos como a proteção antioxidante e a degradação proteica. Um outro resultado novo e importante descrito neste capítulo foi a dependência da assinatura metabólica em relação ao tipo histológico do tumor. Enquanto as principais alterações observadas nos adenocarcinomas (AdC) se relacionaram com o metabolismo fosfolipídico e proteico, os carcinomas de células escamosas (SqCC) apresentaram perfis glicolíticos e glutaminolíticos mais pronunciados, sendo possível construir um modelo válido para a discriminação destes subtipos. No Capítulo 4, apresenta-se o estudo metabolómico por RMN de plasma sanguíneo de mais de 100 doentes e quase 100 controlos saudáveis, do qual resultou um modelo multivariado com uma taxa de classificação de 87%. A distinção entre os grupos foi feita essencialmente com base nos níveis de lactato, piruvato, acetoacetato, lipoproteínas LDL+VLDL e glicoproteínas (aumentados nos doentes), juntamente com os níveis de glutamina, histidina, valina, metanol, lipoproteínas HDL e dois compostos não identificados (diminuídos nos doentes). Estas variações foram detetadas desde os estádios iniciais da doença e a magnitude de algumas delas dependeu do tipo histológico, embora não permitindo discriminar AdC de SqCC. Para além disso, mostra-se neste capítulo que o desequilíbrio dos grupos controlo e cancro em termos da idade dos indivíduos poderá ter alguma influência nos resultados, e apresenta-se uma tentativa exploratória de validação externa, que resultou numa taxa de classificação de 85%. O estudo por RMN do perfil metabólico da urina dos doentes com cancro do pulmão e dos controlos é apresentado no Capítulo 5. Comparativamente ao plasma, o modelo construído com os perfis urinários apresentou uma taxa de classificação superior (97%). Após uma avaliação cuidada da possível influência do género, idade e hábitos tabágicos, um conjunto de 19 metabolitos foi proposto como estando relacionado com a doença (incluindo 3 compostos desconhecidos e 6 parcialmente identificados como metabolitos N-acetilados). Tal como no caso do plasma, estas variações foram detetadas em doentes no estádio inicial e mostraram alguma dependência em relação ao tipo histológico, obtendo-se um modelo válido para a discriminação AdC vs. SqCC, ainda que com um poder preditivo modesto. Para além disso, o teste preliminar de validação externa revelou 100% de sensibilidade e 90% de especificidade, o que é um resultado bastante promissor em termos da potencial utilização dos perfis urinários em aplicações clínicas futuras. No Capitulo 6, descreve-se a caracterização dos perfis metabólicos da urina (de um subgrupo de indivíduos) por cromatografia líquida de ultra-eficiência acoplada a espetrometria de massa (UPLC-MS). Embora não avançando muito na identificação estrutural de possíveis marcadores, este estudo reforçou o valor diagnóstico da urina, já que os modelos multivariados resultantes apresentaram taxa de classificação e poder preditivo elevados. Finalmente, no Capítulo 7, apresentam-se as principais conclusões deste trabalho, realçando o contributo da metabolómica integrada de tecidos e biofluidos para a compreensão do metabolismo alterado do cancro do pulmão e para a deteção de novos perfis marcadores com valor diagnóstico.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2014
Endometriosis is characterized by the presence of endometriotic epithelium and stroma in ectopic locations. It has been proposed that this tissue can synthesize its own growth-supporting steroid hormones by collecting substrate steroids from blood supply or by using cholesterol. In the present work Trilostane, an 3β-hydroxysteroid dehydrogenase inhibitor, was tested. The analytical part was based on High pressure Liquid Chromatography-tandem Mass Spectrometry analysis with a reverse-phase column coupled to an electrospray source to analyse steroid concentrations. As steroids require the overcome of some difficulties in terms of analysis, oxime derivatization was the key in the analytical method in order to obtain reliable results. To investigate androgen metabolism in endometriosis, endometrial and endometriotic tissues were treated with dehydroepiandrosterone and trilostane, and the steroid concentration in the surrounding medium was measured. Only three compounds were detected in the samples, Testosterone, Dehydroepiandrosterone and androstenedione, while the others were under the lowest limit of quantitation. Androstenedione concentration results were conclusive, since its accumulation was inhibited by trilostane, whilst for the rest of the compounds it was impossible to take reasonable conclusions, which demonstrates that the enzyme is essential in the androgen production in endometriosis. No accumulation of androstenedione was seen in the endometrium tissue.
University of Eastern Finland, Department of Pharmacy.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Ivo Vrobel Supervisors: Prof. RNDr. Petr Solich, CSc; Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague Prof. Seppo Auriola, MSc.(Chem.) Marko Lehtonen; Department of Pharmaceutical Chemistry, School of Pharmacy, University of Eastern Finland in Kuopio Title of master's thesis: LC-MS/MS analysis of acetylcholine in brain microdialysis samples Novel fast and simple LC-MS/MS method of ACh quantification in brain microdialysis samples utilizing stable-isotope-labeled IS was developed. The chromatographic step is based on revered-phase mode of pentafluorophenylpropyl (PFPP) column. The satisfactory retention of ACh is achieved with highly aqueous mobile phase containing 0.05% of the ion-pairing agent TFA and 4% of ACN in 4 min analytical run. Ionization of ACh and IS with low background noise and tolerant towards use of TFA was performed with atmospheric pressure thermospray ionization (APTSI). The selectivity of ACh and IS detection was obtained by SRM modes of MS/MS in the linear ion trap mass analyzer. The performance of developed method was cross validated to the validated method used in the laboratory for ACh measurements. The set of microdialysis...

LC-MS of protein drugs requires new ideas in bonded phase design rather than adapting bonded phases from the realm of small-molecule drugs. The polymer-shell bonded phase is designed to interact with larger molecules and to shield proteins from the silica substrate. The particles consist of a core of solid silica and a shell of dense polymer brush. The polymer layer is thick enough to protect the protein from interactions with silanols to reduce peak tailing. The polymer contains multiple functional groups that introduce more selectivity. This design gives unprecedented LC resolution and MS sensitivity. Our group has developed polymer shell bonded phases for hydrophobic interaction chromatography (HIC-MS) of antibody-drug conjugates (ADCs), hydrophilic interaction liquid chromatography (HILIC-MS) of glycoproteins, and reversed-phase liquid chromatography (RPLC-MS) of monoclonal antibodies. Since HIC is not in-line compatible with MS due to the high salt levels, it is laborious to identify the constituents of HIC peaks. An MS-compatible alternative to HIC is reported here: native reversed phase liquid chromatography (nRPLC). This employs a mobile phase 50 mM ammonium acetate for high sensitivity in MS, and elution with a gradient of water/isopropanol. The nRPLC-MS data show that all ADC species, ranging from drug-to-antibody ratios of 1 to 8, remained intact and native on the column. As we adapt this concept to intact proteins, we find that lysozyme and α-chymotrypsinogen A are both eluted in their native conformations. We also use the polymer-shell concept to resolve IgG1 free thiol variants by RPLC-MS with 0.5% formic acid. Since there are always other variants besides the intended ones, the need for high MS sensitivity is desired to distinguish subtle mass change between disulfide bond and free thiols. Overall, MS sensitivity increases 10X relative while all of the thiol variants are well resolved by the polymethylmethacrylate bonded phase.

Modern separation and spectrometric techniques for biological sample lipidom investigation Due recent progress in field of mass spectrometry the lipidomics, part of metabolomics, is increasing its importance for broad fields of biological study. The aim of this study is to test the lipid extraction techniques and to optimize the preseparation and separation of lipids suitable for mass spectrometry detection. The fragmentation patterns of four, the most abundant lipid classes of glycerolipids (PC, PE, TG, DG), were acquired for the proposed system. These patterns were compared with literature. The most appropriate method for extraction was declared technique according Folch based on methanol and chloroform solution. The preseparation due SPE method is very useful tool for lipid determination. The optimized were focused to reach higher recovery especially in polar lipid fraction. Proposed HPLC system is based on methanol with ammonium buffer, water and isopropanol. The testing was done on three columns with different type of sorbents (Gemini, Syncronis and Kinetex). The separation was evaluated according mass spectrometer response, shape and wide of particular analytes peaks. Composition contains 20% of water was determinate as the best and also the best separation was achieved by Kinetex column. The proposed...