Estabelecimento do perfil metabolómico volátil da urina e saliva, como estratégia não-invasiva, para a deteção de potenciais biomarcadores de diferentes tipos de cancro

Com este trabalho pretendeu-se estabelecer o perfil metabolómico volátil de amostras de fluidos biológicos, nomeadamente saliva e urina, de pacientes com cancro da mama e do pulmão e de indivíduos saudáveis (grupo controlo), utilizando a Microextração em Fase Sólida em modo headspace (HS-SPME) seguida de Cromatografia Gasosa acoplada à Espectrometria de Massa (GC-MS). Efetuou-se a comparação entre os perfis voláteis dos grupos estudados com o objetivo de identificar metabolitos que possam ser considerados como potenciais biomarcadores dos tipos de cancro em estudo. De modo a otimizar a metodologia extrativa, HS-SPME, foram avaliados os diferentes parâmetros experimentais com influência no processo extrativo. Os melhores resultados foram obtidos com a fibra CAR/PDMS, usando um volume de 2 mL de saliva acidificada, 10% NaCl (m/v) e 45 minutos de extração a uma temperatura de 37±1°C. Para a urina foi utilizada a mesma fibra, 4 mL de urina acidificada, 20% NaCl (m/v) e 60 minutos de extração a 50±1°C. Nas amostras de saliva e urina, foram identificados 243 e 500 metabolitos voláteis respetivamente, sendo estes pertencentes a diferentes famílias químicas. Posteriormente, utilizou-se a análise discriminante por mínimos quadrados parciais (PLS-DA) que permitiu observar uma boa separação entre os grupos controlo e oncológicos. Nas amostras salivares o grupo de pacientes com cancro da mama foi maioritariamente caracterizado pelo metabolito ácido benzeno carboxílico e o grupo de pacientes com cancro do pulmão pelo ácido hexanóico. Na urina o grupo de pacientes com cancro da mama foi maioritariamente caracterizado pelo metabolito 1-[2-(Isobutiriloxi)-1-metiletil]-2,2-dimetilpropil 2-metilpropanoato e o grupo de pacientes com cancro do pulmão pelo o-cimeno. Além da metodologia PLS-DA foi realizada a validação cruzada de monte carlo (MCCV) tendo-se obtido uma elevada taxa de classificação, sensibilidade e especificidade o que demonstra a robustez dos dados obtidos.

Use of novel biomarkers (Homocysteine, vitamin B6, B9 and B12) on the assessing the progression of cardiovascular disease

A large number of evidences correlate elevated levels of homocysteine (Hcys) with a higher cardiovascular diseases (CVDs) risk, especially, atherosclerosis. Similarly, abnormal low levels of the vitamins B6, B9 and B12 are associated to an instability in the methionine cycle with an over production of Hcys. Thus, biomedical sciences are looking forward for a cheaper, faster, precise and accurate analytical methodology to quantify these compounds in a suitable format for the clinical environment. Therefore the objective of this study was the development of a simple, inexpensive and appropriate methodology to use at the clinical level. To achieve this goal, a procedure integrating a digitally controlled (eVol®) microextraction by packed sorbent (MEPS) and an ultra performance liquid chromatography (UPLC) coupled to a photodiode array detector (PDA) was developed to identify and quantify Hcys vitamins B6, B9 and B12. Although different conditions were assayed, we were not able to combine Hcys with the vitamins in the same analytical procedure, and so we proceeded to the optimization of two methods differing only in the composition of the gradient of the mobile phase and the injected volume. It was found that MEPS did not bring any benefit to the quantification of the Hcys in the plasma. Therefore, we developed and validate an alternative method that uses the direct injection of treated plasma (reduced and precipitated). This same method was evaluated in terms of selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effect and precision (intra-and inter-day) and applied to the determination of Hcys in a group composed by patients presenting augmented CVD risk. Good results in terms of selectivity and linearity (R2> 0.9968) were obtained, being the values of LOD and LOQ 0.007 and 0.21 mol / L, respectively. The intra-day precision (1.23-3.32%), inter-day precision (5.43-6.99%) and the recovery rate (82.5 to 93.1%) of this method were satisfactory. The matrix effect (>120%) was, however, higher than we were waiting for. Using this methodology it was possible to determine the amount of Hcys in real plasma samples from individuals presenting augmented CVD risk. Regarding the methodology developed for vitamins, despite the optimization of the extraction technique and the chromatographic conditions, it was found that the levels usually present in plasma are far below the sensitivity we obtained. Therefore, further optimizations of the methodology developed are needed. As conclusion, part of the objectives of this study was achieved with the development of a quick, simple and cheaper method for the quantification of Hcys.

Avaliação de nova estratégia extrativa, baseada em NTDs, combinada com dados de análise cromatigráfica e quimiométrica para a deteção de biomarcadores de diferentes tipos de cancro

A elevada incidência e mortalidade mundiais associadas ao cancro justificam o desenvolvimento e implementação de estratégias eficazes e não-invasivas conducentes a um diagnóstico precoce. Neste contexto, pretendeu-se avaliar a performance de uma metodologia inovadora, a microextração por “needle trap” (NTME), na extração de metabolitos voláteis (VOMs) da urina de pacientes oncológicos com diferentes tipos de cancro - cólon, pulmão e mama, e de indivíduos saudáveis, com a finalidade de identificar um conjunto de VOMs potenciais biomarcadores dos diferentes tipos cancros em estudo. De modo a maximizar a eficiência da extração dos VOMs, foram otimizados diferentes parâmetros experimentais, nomeadamente a natureza do sorvente, a temperatura, o tempo de equilíbrio, o volume de headspace, a força iónica, o pH do meio e o volume e a agitação da amostra. Usando como sorvente o DVB/Car1000/CarX, os melhores resultados foram obtidos com 4 mL de urina acidificada (pH= 2), 20% NaCl, 40 mL de headspace e 40 min de equilíbrio a 50 °C. Foi ainda avaliada a estabilidade dos VOMs no sorvente até 72 h após a extração. Nos quatro grupos em estudo foram identificados, por GC-MS, 259 VOMs pertencentes a diversas famílias químicas, nomeadamente cetonas, compostos sulfurados, furânicos e terpénicos. A matriz de dados obtida para cada grupo em estudo foi submetida a análise discriminante, usando o método dos mínimos quadrados parciais (PLS-DA), que resultou em clusters distintos diferenciadores de cada grupo. A aplicabilidade do modelo foi avaliada através do método de classificação SIMCA (modelagem suave e independente de analogias de classe), com elevadas taxas de classificação, sensibilidade e especificidade. Este foi o primeiro estudo usando NTME para o estabelecimento do padrão volatómico da urina. Os resultados obtidos revelam-se muito promissores originando perfis voláteis de maior expressividade, mais completos e abrangentes, que os obtidos usando metodologias de referência.

Plataforma para o suporte de blended peer assisted learning

Este projecto foi realizado na Universidade da Madeira, no âmbito do Mestrado em Engenharia Informática e tem como título “Plataforma para o suporte de Blended Peer Assisted Learning”. Ao longo da nossa pesquisa sobre os vários métodos de aprendizagem online, deparamo-nos com uma grande lacuna que abrange a maior parte dos sistema de ensino assistidos por computador, ou seja, todas elas preocupam-se com a passagem de conhecimentos, mas raras são aquelas que têm em atenção o tipo de utilizador, qual a seu percurso académico e profissional, qual a metodologia que fará com que o mesmo capte melhor os conteúdos, etc. Com este objectivo em mente, e tendo em atenção as diversas plataformas e metodologias de ensino existentes, optou-se por elaborar uma arquitectura de uma plataforma capaz de centralizar na mesma, um conjunto de funcionalidades e metodologias que possibilitassem um acompanhamento mais específico do utilizador, proporcionando um maior conhecimento, através do qual poderia encaminhar o utilizador para a estratégia de aprendizagem que mais se adequasse a um utilizador com as suas características. Como se poderá constatar no decorrer desta dissertação, a plataforma desenhada e o módulo desenvolvido têm como base teórica o Peer Assisted Learning (PAL) e as suas estratégias de aprendizagem. O PAL é um conceito relativamente novo, que se encontra em plena ascensão, sendo cada vez maior o número de instituições/organizações que adoptam o PAL como metodologia de ensino para a formação dos seus membros. Este crescimento deve-se em grande parte às várias estratégias PAL que visam uma maior adequação ao tipo de utilizador, contribuindo assim para uma aprendizagem mais rápida e eficaz. Uma vez que a implementação da plataforma na sua totalidade seria de todo impossível, optouse por desenvolver apenas um dos módulos referente à estratégia de PAL – Peer Tutoring (PT). No final, o objectivo principal, passa não só por fornecer as bases necessárias ao desenvolvimento da referida plataforma, mas também pela disponibilização do módulo de PT que servirá de referência para o desenvolvimento das restantes estratégias. Tendo em atenção toda a investigação efectuada, facilmente se depreende as inúmeras vantagens que podem advir da utilização do PAL, das quais se salienta, a adequação da estratégia PAL mais indicada para cada tipo de utilizador.

Effectiveness of different solid-phase microextraction fibres for differentiation of selected Madeira island fruits based on their volatile metabolite profile: identification of novel compounds

A headspace solid-phase microextraction (HS-SPME) procedure based on five commercialised fibres (85 μm polyacrylate – PA, 100 μm polydimethylsiloxane – PDMS, 65 μm polydimethylsiloxane/divinylbenzene – PDMS/DVB, 70 μm carbowax/divinylbenzene – CW/DVB and 85 μm carboxen/polydimethylsiloxane – CAR/PDMS) is presented for the characterization of the volatile metabolite profile of four selected Madeira island fruit species, lemon (Citrus limon), kiwi (Actinidia deliciosa), papaya (Carica papaya L.) and Chickasaw plum (Prunus angustifolia). The isolation of metabolites was followed by thermal desorption gas chromatography–quadrupole mass spectrometry (GC–qMS) methodology. The performance of the target fibres was evaluated and compared. The SPME fibre coated with CW/DVB afforded the highest extraction efficiency in kiwi and papaya pulps, while in lemon and plum the same was achieved with PMDS/DVB fibre. This procedure allowed for the identification of 80 compounds, 41 in kiwi, 24 in plums, 23 in papaya and 20 in lemon. Considering the best extraction conditions, the most abundant volatiles identified in kiwi were the intense aldehydes and ethyl esters such as (E)-2-hexenal and ethyl butyrate, while in Chicasaw plum predominate 2-hexenal, 2-methyl-4-pentenal, hexanal, (Z)-3-hexenol and cyclohexylene oxide. The major compounds identified in the papaya pulp were benzyl isothiocyanate, linalool oxide, furfural, hydroxypropanone, linalool and acetic acid. Finally, lemon was shown to be the most divergent of the four fruits, being its aroma profile composed almost exclusively by terpens, namely limonene, γ-terpinene, o-cymene and α-terpinolene. Thirty two volatiles were identified for the first time in the fruit or close related species analysed and 14 volatiles are reported as novel volatile metabolites in fruits. This includes 5 new compounds in kiwi (2-cyclohexene-1,4-dione, furyl hydroxymethyl ketone, 4-hydroxydihydro-2(3H)-furanone, 5-acetoxymethyl-2-furaldehyde and ethanedioic acid), 4 in plum (4-hydroxydihydro-2(3H)-furanone, 5-methyl-2-pyrazinylmethanol, cyclohexylene oxide and 1-methylcyclohexene), 4 in papaya (octaethyleneglycol, 1,2-cyclopentanedione, 3-methyl-1,2-cyclopentanedione and 2-furyl methyl ketone) and 2 in lemon (geranyl farnesate and safranal). It is noteworthy that among the 15 volatile metabolites identified in papaya, 3-methyl-1,2-cyclopentanedione was previously described as a novel PPARγ (peroxisome proliferator-activated receptor γ) agonist, having a potential to minimize inflammation.

HPLC-DAD methodology for the quantification of organic acids, furans and polyphenols by direct injection of wine samples

This article proposes a simple and sensitive HPLC method with photo-diode array detection for the analysis of organic acids, monomeric polyphenols and furanic compounds in wine samples by direct injection. The chromatographic separation of 8 organic acids, 2 furans and 22 phenolic compounds was carried out with a buffered solution (pH 2.70) and acetonitrile as mobile phases and a difunctionally bonded C18 stationary phase, Atlantis dC18 (250 4.6 mm, 5mm) column. The elution was performed in 12 min for the organic acids and in 60 min for the phenolic compounds, including phenolic acids, stilbenes and flavonoids. Target compounds were detected at 210 nm (organic acids, flavan-3-ols and benzoic acids), 254 nm (ellagic acid), 280 nm (furans and cinnamic acid), 315 nm (hydroxycinnamic acids and trans-resveratrol) and 360 nm (flavonoids). The RSD for the repeatability test (n55) of peak area and retention times were below 3.1 and 0.3%, respectively, for phenolics and below 1.0 and 0.2% for organic acids. The RSDs expressing the reproducibility of the method were higher than for the repeatability results but all below 9.0%. Method accuracy was evaluated by the recovery results, with averaged values between 80 and 104% for polyphenols and 97–105% for organic acids. The calibration curves, obtained by triplicate injection of standard solutions, showed good linearity with regression coefficients higher than 0.9982 for polyphenols and 0.9997 for organic acids. The LOD was in the range of 0.07–0.49 mg/L for polyphenols (cinnamic and gallic acids, respectively) and 0.001–0.046 g/L for organic acids (oxalic and lactic acids, respectively). The method was successfully used to measure and assess the polyphenolic fingerprint and organic acids profile of red, white, rose ´ and fortified wines.

Dynamic headspace solid-phase microextraction combined with one-dimensional gas chromatography–mass spectrometry as a powerful tool to differentiate banana cultivars based on their volatile metabolite profile

In this study the effect of the cultivar on the volatile profile of five different banana varieties was evaluated and determined by dynamic headspace solid-phase microextraction (dHS-SPME) combined with one-dimensional gas chromatography–mass spectrometry (1D-GC–qMS). This approach allowed the definition of a volatile metabolite profile to each banana variety and can be used as pertinent criteria of differentiation. The investigated banana varieties (Dwarf Cavendish, Prata, Maçã, Ouro and Platano) have certified botanical origin and belong to the Musaceae family, the most common genomic group cultivated in Madeira Island (Portugal). The influence of dHS-SPME experimental factors, namely, fibre coating, extraction time and extraction temperature, on the equilibrium headspace analysis was investigated and optimised using univariate optimisation design. A total of 68 volatile organic metabolites (VOMs) were tentatively identified and used to profile the volatile composition in different banana cultivars, thus emphasising the sensitivity and applicability of SPME for establishment of the volatile metabolomic pattern of plant secondary metabolites. Ethyl esters were found to comprise the largest chemical class accounting 80.9%, 86.5%, 51.2%, 90.1% and 6.1% of total peak area for Dwarf Cavendish, Prata, Ouro, Maçã and Platano volatile fraction, respectively. Gas chromatographic peak areas were submitted to multivariate statistical analysis (principal component and stepwise linear discriminant analysis) in order to visualise clusters within samples and to detect the volatile metabolites able to differentiate banana cultivars. The application of the multivariate analysis on the VOMs data set resulted in predictive abilities of 90% as evaluated by the cross-validation procedure.

A fast method using a new hydrophilic–lipophilic balanced sorbent in combination with ultra-high performance liquid chromatography for quantification of significant bioactive metabolites in wines

This manuscript describes the development and validation of an ultra-fast, efficient, and high throughput analytical method based on ultra-high performance liquid chromatography (UHPLC) equipped with a photodiode array (PDA) detection system, for the simultaneous analysis of fifteen bioactive metabolites: gallic acid, protocatechuic acid, (−)-catechin, gentisic acid, (−)-epicatechin, syringic acid, p-coumaric acid, ferulic acid, m-coumaric acid, rutin, trans-resveratrol, myricetin, quercetin, cinnamic acid and kaempferol, in wines. A 50-mm column packed with 1.7-μm particles operating at elevated pressure (UHPLC strategy) was selected to attain ultra-fast analysis and highly efficient separations. In order to reduce the complexity of wine extract and improve the recovery efficiency, a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a new macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis™ HLB), was performed prior to UHPLC–PDA analysis. The calibration curves of bioactive metabolites showed good linearity within the established range. Limits of detection (LOD) and quantification (LOQ) ranged from 0.006 μg mL−1 to 0.58 μg mL−1, and from 0.019 μg mL−1 to 1.94 μg mL−1, for gallic and gentisic acids, respectively. The average recoveries ± SD for the three levels of concentration tested (n = 9) in red and white wines were, respectively, 89 ± 3% and 90 ± 2%. The repeatability expressed as relative standard deviation (RSD) was below 10% for all the metabolites assayed. The validated method was then applied to red and white wines from different geographical origins (Azores, Canary and Madeira Islands). The most abundant component in the analysed red wines was (−)-epicatechin followed by (−)-catechin and rutin, whereas in white wines syringic and p-coumaric acids were found the major phenolic metabolites. The method was completely validated, providing a sensitive analysis for bioactive phenolic metabolites detection and showing satisfactory data for all the parameters tested. Moreover, was revealed as an ultra-fast approach allowing the separation of the fifteen bioactive metabolites investigated with high resolution power within 5 min.

Quantification of polyphenols with potential antioxidant properties in wines using reverse phase HPLC

A RP-HPLC method with photodiode array detection (DAD) was developed to separate, identify and quantify simultaneously the most representative phenolic compounds present in Madeira and Canary Islands wines. The optimized chromatographic method was carefully validated in terms of linearity, precision, accuracy and sensitivity. A high repeatability and a good stability of phenolics retention times (a3%) were obtained, as well as relative peak area. Also high recoveries were achieved, over 80.3%. Polyphenols calibration curves showed a good linearity (r2 A0.994) within test ranges. Detection limits ranged between 0.03 and 11.5 lg/mL for the different polyphenols. A good repeatability was obtained, with intra-day variations less than 7.9%. The described method was successfully applied to quantify several polyphenols in 26 samples of different kinds of wine (red, ros and white wines) from Madeira and Canary Islands. Gallic acid was by far the most predominant acid. It represents more than 65% of all phenolics, followed by p-coumaric and caffeic acids. The major flavonoid found in Madeira wines was trans-resveratrol. In some wines, (–)-epicatechin was also found in highest amount. Canary wines were shown to be rich in gallic, caffeic and p-coumaric acids and quercetin.

Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldeyde derivatization procedure

This work presents a RP-HPLC method for the simultaneous quantification of free amino acids and biogenic amines in liquid food matrices and the results of the application to honey and wine samples obtained from different production processes and geographic origins. The developed methodology is based on a pre-column derivatization with o-phthaldialdehyde carried out in the sample injection loop. The compounds were separated in a Nova-Pack RP-C18 column (150 mm × 3.9 mm, 4 μm) at 35 °C. The mobile phase used was a mixture of phase A: 10 mM sodium phosphate buffer (pH 7.3), methanol and tetrahydrofuran (91:8:1); and phase B: methanol and phosphate buffer (80:20), with a flow rate of 1.0 ml/min. Fluorescence detection was used at an excitation wavelength of 335 nm and an emission wavelength of 440 nm. The separation and quantification of 19 amino acids and 6 amines was carried out in a single run as their OPA/MCE derivatives elute within 80 min, ensuring a reproducible quantification. The method showed to be adequate for the purpose, with an average RSD of 2% for the different amino acids; detection limits varying between 0.71 mg/l (Asn) and 8.26 mg/l (Lys) and recovery rates between 63.0% (Cad) and 98.0% (Asp). The amino acids present at the highest concentration in honey and wine samples were phenylalanine and arginine, respectively. Only residual levels of biogenic amines were detected in the analysed samples.

A prática pedagógica com a mediação de filmes e documentários na formação técnica de alunos do Curso Técnico em Edificações do IFPE, Campus Pesqueira, Pernambuco, Brasil

Neste estudo, visamos investigar a inovação pedagógica na formação dos alunos do Curso Técnico em Edificações do Instituto Federal de Educação, Ciência e Tecnologia - Campus Pesqueira – Pesqueira - Pernambuco – Brasil, tendo como objetivo analisar a formação técnica a partir da cultura visual e dos recursos midiáticos atrelados ao mundo concreto da docência escolar. Para tanto, realizamos uma pesquisa qualitativa de cunho etnográfico para capturar o fenômeno estudado, utilizando como instrumentos e técnicas a observação participante, diário de campo, entrevistas, fotografias, filmes e documentários. Dessa forma, coletamos e analisamos os achados, tendo como pano de fundo a cultura das imagens fincada no chão da sala de aula, numa prática pedagógica construcionista inovadora.

A micro-extraction technique using a new digitally controlled syringe combined with UHPLC for assessment of urinary biomarkers of oxidatively damaged DNA

The formation of reactive oxygen species (ROS) within cells causes damage to biomolecules, including membrane lipids, DNA, proteins and sugars. An important type of oxidative damage is DNA base hydroxylation which leads to the formation of 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodG) and 5-hydroxymethyluracil (5-HMUra). Measurement of these biomarkers in urine is challenging, due to the low levels of the analytes and the matrix complexity. In order to simultaneously quantify 8-oxodG and 5-HMUra in human urine, a new, reliable and powerful strategy was optimised and validated. It is based on a semi-automatic microextraction by packed sorbent (MEPS) technique, using a new digitally controlled syringe (eVolH), to enhance the extraction efficiency of the target metabolites, followed by a fast and sensitive ultrahigh pressure liquid chromatography (UHPLC). The optimal methodological conditions involve loading of 250 mL urine sample (1:10 dilution) through a C8 sorbent in a MEPS syringe placed in the semi-automatic eVolH syringe followed by elution using 90 mL of 20% methanol in 0.01% formic acid solution. The obtained extract is directly analysed in the UHPLC system using a binary mobile phase composed of aqueous 0.1% formic acid and methanol in the isocratic elution mode (3.5 min total analysis time). The method was validated in terms of selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), extraction yield, accuracy, precision and matrix effect. Satisfactory results were obtained in terms of linearity (r2 . 0.991) within the established concentration range. The LOD varied from 0.00005 to 0.04 mg mL21 and the LOQ from 0.00023 to 0.13 mg mL21. The extraction yields were between 80.1 and 82.2 %, while inter-day precision (n=3 days) varied between 4.9 and 7.7 % and intra-day precision between 1.0 and 8.3 %. This approach presents as main advantages the ability to easily collect and store urine samples for further processing and the high sensitivity, reproducibility, and robustness of eVolHMEPS combined with UHPLC analysis, thus retrieving a fast and reliable assessment of oxidatively damaged DNA.

Caracterização aromática de castas produtoras de vinho Madeira: boal, malvazia, sercial e verdelho

Maria Arminda Costa Alves e José Carlos Antunes Marques

O percurso curricular alternativo, um desafio à inovação pedagógica?: uma abordagem etnográfica aos cenários de aprendizagem de uma turma de 5º ano com proposta de PCA

Este estudo pretendeu descrever, compreender e interpretar a cultura emergente de uma turma de 5º Ano de Escolaridade com proposta de Percurso Curricular Alternativo (PCA) em que se procurou esclarecer, à luz do conceito de inovação de que forma o PCA se constitui um desafio à Inovação Pedagógica. Ao debruçar-se sobre os padrões culturais da turma procura conhecer e avaliar o impacto desta proposta alternativa, na vida dos alunos, através do conhecimento das representações de todos os envolvidos no projeto. Além de pretender situar as práticas pedagógicas em termos de inovação ou o contínuo de práticas tradicionais, procura compreender que ambientes são emergentes da utilização das TIC. A presente investigação insere-se numa abordagem metodológica de natureza qualitativa, de cariz etnográfico, justificada pela natureza do estudo. Desenvolve-se através da imersão da investigadora no ambiente natural dos sujeitos, com vista à descrição pormenorizada e facetada da vida do grupo e o acesso a formas de entendimento e compreensão da realidade estudada a partir dos padrões culturais e significados vividos no interior da turma. Foram utilizadas diversas formas de recolha de dados, com destaque para a observação participante e a entrevista, que constituíram os principais recursos da investigação empírica, ainda que complementada com registos de cariz etnográfico como notas de campo, conversas informais e dados de opinião, recolhidos durante a permanência no contexto do estudo. As conclusões desta investigação apontam para o reconhecimento do PCA como uma medida positiva para o aluno na construção do seu projeto de vida pessoal, valorização, integração social e profissional, plenas. A utilização da tecnologia permitiu instituir novos contextos de aprendizagem ao nível micro, da sala de aula e romper com princípios, crenças e atitudes estruturantes da escola tradicional, prefigurando um desafio à Inovação Pedagógica, ou seja, à mudança e transformação da escola.

Evolution of ethyl carbamate during Madeira wine ageing by GC-MS: a new methodology

Recently, ethyl carbamate (EC) was reclassified by the International Agency for Research on Cancer (IARC) as "probably carcinogenic to humans" and occurs mainly in fermented beverages. Nowadays many countries have set limit values for EC in alcoholic beverages. In this sense and taking into account the low concentrations found in alcoholic beverages, the scientific community has shown interest for the development of new analytical methods, whereby its simplification plays an important role in the EC control and prevention. Firstly, a simple, rapid and sensitive methodology was developed for the EC quantification in fortified wines by microextraction by packed sorbent (MEPS) with gas chromatography coupled with a mass spectrometer detector (GC-MS). This method showed good linearity (R2 = 0.999) and sensitivity (LOD = 1.5 μg/L). The accuracy of the method was assessed by means of repeatability and reproducibility (RSD < 7%). Moreover, a good recovery has been demonstrated (97 – 106%) as well as its applicability (16 fortified wines). Thus, the developed methodology has proven to be an excellent approach for routine quantification of EC in fortified wines. The EC evolution was also evaluated during a year and half of Madeira wine ageing submitted to two traditional ageing methods, estufagem and canteiro, in order to evaluate the formation kinetic. The results revealed that estufagem process increased the formation kinetic and promoted a linear increase of the EC concentration (R2 ≥ 0.977), proportionally to the ageing time (4 months). However, when the wines are firstly submitted to estufagem and then undergo canteiro ageing, the EC values remain almost constant during the following 14 months. The results suggest that estufagem does not seem to be the critical factor in the EC formation, but instead the amount of precursors in the medium.