Mycotoxins are secondary metabolites of moulds, which attack cereals, for example barley, from which mycotoxins then get to beer. This submitted work is focused on ochratoxin A, deoxynivalenol and zearalenone, which can occur in beer. The first part of this master’s thesis consists of literary research, which describes mycotoxins in general, points out their occurrence, prevention of their formation and delivers information about their physical and chemical properties and toxicity. Furthermore, the research contains basis of malt and beer technology, the occurrence of mycotoxins in beer and raw materials for its production. The research describes changes in concentration of mycotoxins across malt and beer production. The next part deals with possibilities of determination of mycotoxins in barley, malt and beer, compares individual methods of their determination and points out many difficulties of some analyses. The experimental part of this work pursues determination of ochratoxin A, deoxynivalenol and zearalenone in different types of beer with the help of UPLC-FLR, HPLC-MS and ELISA. Instrumental techniques are validated and gathered results are compared with the results in literature. The goal of this master’s thesis is to assess the stability of ochratoxin A and deoxynivalenol in beer over time. The gained results show that there are changes in the concentration of ochratoxin A over time, nevertheless those changes show no pattern. Overall, there was a decrease in concentration in 47 % of the samples and an increase in 28 % of them. In the rest of the samples the concentration did not change. The concentration of deoxynivalenol does not change over time. One of the other goals of this thesis is monitoring of selected mycotoxins in beer. The average concentration of ochratoxin A in the samples was 39 ng/l and deoxynivalenol 9,9 g/l. Zearalenone did not occur in any of the samples when determined by liquid chromatography. All results agree with literature. Next, the thesis compares different analytical methods for determination of ochratoxin A, deoxynivalenol and zearalenone. The screening method ELISA is compared to UPLC-FLR and HPLC-MS. The determination of ochratoxin A by ELISA has shown to be time consuming, nevertheless the results responded to instrumental technique. ELISA overestimated the results of determination of deoxynivalenol in beer by 363–697 % and with zearalenone there were found false positive results.

This diploma thesis deals with the development and validation of an analytical method using high performance liquid chromatography method with fluorescence detection for the simultaneous determination of aflatoxins and ochratoxin A in herbal and fruit tea. The theoretical part describes the most common groups of mycotoxins and the most important methods for their determination in food. The great attention is devoted to HPLC method and the overview of the derivatization techniques for aflatoxins B1 and G1 fluorescence response enhancement. The practical part of this study focuses on the optimization of sample extraction and purification, the settings of the instrumental analysis and the photochemical reactor assembly. The thesis also involves the determination of the basic performance characteristics for the successful method validation.

Ochratoxin A (OTA) is a potent mycotoxin found in a wide range of agricultural products that has been linked to mitochondrial damage and renal disease. The standard methods for OTA analysis currently rely on the use of high-performance liquid chromatography (HPLC) coupled to fluorescence detection or mass spectrometry. Toward a highthroughput analysis of OTA, a single-stranded DNA aptamer, modified with a fluorophore, coupled to a complementary sequence, modified with a FRET-based quencher that dissociates in the presence of the target toxin, is proposed. In order to integrate “target trapping,” aptamer immobilization methods were explored to mediate interference issues. Assays were evaluated using wine and blood serum matrices. A solution-based assay in a 96-well plate format provided a limit-of-detection of 2.7 ng/mL which would be suitable for many of the proposed applications. Immobilized aptamer formats, however, were not reliable, and a range of limitations to applications of the assay were identified.

L'ochratoxine A (OTA), principalement produite dans le café par les moisissures A. ochraceus et A. westerdijkiae, suscite une attention particulière pour ses effets néphrotoxiques, immunotoxiques, tératogènes et cancérigènes. La présence d'OTA dans les fèves de café peut être mise en relation avec les conditions de récolte, les conditions de traitement post-récolte et les conditions de stockage et de transport. Dans certains pays producteurs, les dommages causés sur les grains de café par des espèces fongiques sont liés à des teneurs élevées en OTA. La dynamique et la biodiversité des populations microbiennes (bactéries, levures, moisissures) lors des traitements post-récolte du café a été étudiée par une méthode d'analyse moléculaire globale des flores, la PCR-DGGE. Il a été observé une évolution et une diversité des flores microbiennes en fonction du type et des étapes des traitements utilisés, spécifiques des lieux de production. La région génomique ciblée et la proximité phylogénétique sont un obstacle à l'identification des souches ochratoxinogènes par l'approche globale utilisée. De plus, une méthode simple et rapide de différenciation moléculaire d'Aspergillus westerdijkiae et d'Aspergillus ochraceus a été mise au point et couplée avec l'analyse d'image pour permettre la « quantification » d'Aspergillus westerdijkiae. Des phénomènes de compétition/inhibition de la croissance et production d'OTA (supérieurs à 90%) ont été mis en évidence pour des souches d'Aspergillus niger et d'Aspergillus ochraceus faiblement productrices d'OTA vis-à-vis d'Aspergillus westerdijkiae qui est l'une des espèces les plus fortement productrices de la mycotoxine sur café. Les résultats obtenus au cours de ce travail sont importants pour l'amélioration des connaissances sur la dynamique des populations microbiennes au cours des procédés de transformation du café ainsi que pour de possibles applications en prévention et maîtrise de la contamination du café par l'OTA. / Ochratoxin A (OTA) is mainly produced on coffee beans by fungal species Aspergillus ochraceus and Aspergillus westerdijkiae, and is known for its impact on human health through nephrotoxic, immunotox, teratogenic and oncogenic effects.The OTA content in coffee was shown to be closely linked to harvesting conditions, post-harvest processing conditions and especially dry processing, storage and transportation conditions. In some producing countries, damaged caused on beans by fungal communities undoubtedly lead to high OTA contents in coffee. In order to understand the OTA contamination process, the dynamics and biodiversity of microbial populations (bacteria, yeast and moulds) was analyzed during post-harvest treatment by use of a global microbial ecology approach at the molecular level, so-called PCR-DGGE. Specific variations in evolution and diversity of microbial flora were observed as a function of the step and type of treatment, which were specific of the location of production. The genomic region targeted by the global approach and the genetic proximity of ochratoxigenic fungal strains made their study and identification difficult using the the global approach. In addition, a simple and rapid method for the molecular differentiation of A. westerdijkiae and A. ochraceus was established and, coupled with image analysis, allowed the quantification of A. westerdijkiae.Moreover, competition and inhibition effects on growth and OTA production (>90%) could be observed for low OTA producers A. niger and A. ochraceus species towards the high OTA producer A. westerdijkiae species. Results obtained during this study are of importance for understanding microbial population dynamics during coffee transformation processes. Moreover, it provides possible clues for prevention and control of coffee contamination by OTA.

Tout au long de la chaîne alimentaire, des moisissures peuvent se développer et produire des mycotoxines. Ce sont des composés toxiques naturels issus du métabolisme secondaire des moisissures, susceptibles de contaminer l'alimentation animale et humaine, provoquant de nombreuses pathologies (hépatotoxicité, néphrotoxicité, neurotoxicité, mutagénicité, tératogénicité, cancérogénicité,…). La première étape de ce travail était d'évaluer la présence simultanée de l'ochratoxine A (OTA), de la citrinine (CIT), des aflatoxines (AFs), de la zéaralénone (ZEA), de la fumonisine (FB) et des trichothécènes dans des aliments destinés aux humaines (céréales, lait, café, jambon) et aux animaux (croquettes de chat et chien, foins). En général plusieurs mycotoxines coexistaient. Certains échantillons pour les humains dépassaient les limites autorisées en mycotoxines dans l'Union Européenne. Suite à l'étude de simulation d'apport en mycotoxines dans une ration quotidienne, nous avons constaté que les doses journalières admissibles (DJA) peuvent être dépassées. La deuxième phase consistait à étudier l'impact des mycotoxines seules ou en combinaison sur la viabilité cellulaire et la génotoxicité sur des modèles cellulaires (cellules rénales d'opossum (OK), cellules rénales humaines (HK2), cellules humaines de glandes mammaires (MCF7)) et chez des animaux (porc, rat). Nous avons montré que la CIT, la FB1 et la ZEA agissent en synergie sur la génotoxicité de l'OTA. Chez les animaux, nous avons montré qu'à des doses (5 ng d'OTA/kg poids corporel/ jour et de 200ng FB1/kg pc/j) correspondantes aux DJA, il y avait des effets génotoxiques (formation d'adduits à l'ADN). Nous avons mis en évidence l'implication des mycotoxines dans l'alimentation animale sur la baisse de fertilité et la tératogénicité chez les chats, ainsi que sur la mort des chevaux. Au cours de la troisième partie de cette étude, nous avons testé sur des cultures cellulaires (HK2 et MCF7) et in vivo (poulet) l'effet protecteur du glutathion (GSH) et de la sélénométhionine (SeMet) contre l'OTA responsable de cancers de voie urinaire et la ZEA responsable de baisse de fertilité. Le GSH est un puisant antioxydant et le sélénium est un oligoélément indispensable qui intervient comme co-facteur de nombreuses enzymes ayant des propriétés antioxydantes, comme les glutathion peroxydases. D'une manière générale, au niveau des cellules rénales, le GSH seul et la levure correspondante ont un effet bénéfique vis-à-vis de la génotoxicité de l'OTA ; par contre la sélénométhionine et la levure séléniée augmentent la génotoxicité de l'OTA et de la ZEA. Dans les cellules des glandes mammaires, il y a une nette amélioration vis-à-vis de la génotoxicité des deux mycotoxines lorsque les cellules sont exposées à une seule mycotoxine simultanément au GSH, à la sélénométhionine et aux levures enrichies. Chez les poulets, la diminution de la génotoxicité n'est pas exclusivement corrélée à la capacité des parois de levure ou des levures à adsorber l'OTA. Ces dérivés de levure ont gardé la propriété de partiellement métaboliser l'OTA dans l'intestin. Les parois de levures et les levures enrichies en GSH ont un meilleur pouvoir protecteur que celles enrichies en SeMet / Throughout the food chain, mold can grow and produce mycotoxins. These are toxic compounds "natural" from the secondary metabolism of molds that may contaminate the feed and food, causing many diseases (hepatotoxicity, nephrotoxicity, neurotoxicity, mutagenicity, teratogenicity, carcinogenicity, ...). The first stage of this work was to assess the level of multi-contamination by mycotoxins (OTA, CIT, Afs, ZEA, FB, DON) in food (cereals, milk, coffee, ham) and feed (pet food). Some samples analyzed exceeded the limits of mycotoxins in the European Union. Through the simulation study of mycotoxin intake in a daily diet, we found that the acceptable daily intake (ADI) may be exceeded. The second phase was to study the impact of mycotoxins alone or in combination on cell proliferation, genotoxicity in cellular models (OK, HK2, and MCF7) and animal (pig, rat). We have demonstrated genotoxic effects (formation of DNA adducts) at doses (5 ng OTA / kg bw / day and 200 ng FB1/kg bw / day) considered safe (ADI). We have shown that the CIT, FB1 and ZEA act synergistically on the genotoxicity of OTA. We pointed to the involvement of mycotoxins in animal feed on declining fertility and teratogenicity in cats, as well as the death of horses. In the third part of this study, we tested in cell cultures (HK2 and MCF7) and in vivo (chicken) the protective effect of glutathione (GSH) and selenomethionine (SeMet) against OTA responsible for urinary tract cancers and ZEA reducing fertility. GSH is considered as a potent antioxidant and selenium is a trace essential element that acts as a cofactor of enzymes such glutathione peroxidase. In summary, in kidney cells, GSH and GSH enriched yeast decrease OTA genotoxicity whereas SeMet and SeMet enriched yeast increase genotoxicity of OTA and ZEA. In mammary cells, whatever the compounds gentoxicty of OTA and ZEA significantly decrease. Decrease of OTA genotoxicity in chicken kidney cannot be exclusively explained by adsorption of OTA on yeast by products. The yeast products retain their ability to metabolize the OTA. GSH enriched yeast and yeast cell wells are more efficient than SeMet enriched yeast

As micotoxinas são compostos produzidos por fungos, sendo potencialmente perigosos à saúde humana e animal. A Ocratoxina A (OTA) é uma das micotoxinas mais amplamente estudadas, sendo encontrada em várias matrizes alimentícias. A concentração dessa micotoxina nos alimentos é geralmente muito baixa (da ordem de ng g-1), sendo, portanto, necessário o emprego de técnicas de preparo de amostras que realizem a purificação e pré-concentração no analito. Os métodos de separação e detecção empregados na análise de OTA também devem oferecer sensibilidade adequada para a quantificação do analito. Logo, os objetivos do trabalho foram desenvolver uma metodologia utilizando a técnica in-tube SPME no modo de extração única (flow through extraction) com partículas de C18 como fase extratora e separação e detecção por HPLC-MS/MS. Além disso, otimizar e validar a metodologia proposta e detectar e quantificar a OTA em amostras de vinho. Para isso, a técnica in-tube SPME foi configurada e um tubo de PEEK foi empacotado com partículas de C18, o qual foi utilizado na extração. A otimização do método foi realizada utilizando-se um planejamento experimental composto central 22 + 3 pontos centrais, tendo como fatores a porcentagem de ACN e o tempo durante o carregamento da amostra. A validação da metodologia empregada foi realizada conforme o guia de validação da ANVISA e, posteriormente, amostras de vinho tinto seco e vinho branco seco foram analisadas. O método proposto foi desenvolvido, tendo sua funcionalidade atestada e suas condições de análise melhoradas, sendo utilizado 22 % de ACN e 6 minutos no carregamento da amostra. O método foi validado, mostrando sensibilidade adequada, com limites de detecção e quantificação iguais à 0,02 e 0,05 µg L-1, respectivamente. A linearidade e precisão da metodologia foram avaliadas, apresentando coeficiente de correlação igual a 0,996 e DPR menor que 6%, respectivamente. O método mostrou-se exato nos níveis de concentração médio e alto e a recuperação máxima obtida para o nível alto foi próximo 73%. Amostras brasileiras e estrangeiras de vinho tinto seco e vinho tinto seco branco foram analisadas e a OTA não foi detectada em nenhuma delas. Entretanto, a OTA pode essa estar presente nas amostras analisadas em concentrações mais baixas que as determinadas pelos limites de detecção e quantificação, não sendo potencialmente perigosos à saúde. / Mycotoxins are compounds produced by fungus, being a potential danger to human and animal health. Ochratoxin A (OTA) is a mycotoxin widely studied, being found in a varied of food matrices. OTA concentration in food is generally low (the order of ng g-1), being, therefore, necessary the use of sample preparation techniques to perform the analyte purification and preconcentration in the sample. The separation and detection methods used in OTA analysis also should offer proper sensibility in order to quantify the analyte adequately. In short, the goals of the present work were to develop a methodology using in-tube SPME in flow through extraction mode with C18 particles as extraction phase and separation and detection by HPLC-MS/MS. Further, optimize and validate the proposed methodology and detect and quantify OTA in wine samples. For this, in-tube SPME was configured and a PEEK tube was packed with C18 particles, which was used in the extraction step. The method optimization was achieved using a central composite 22 + 3 central points experimental design, having as factors the percentage of ACN and time during sample loading step. Validation method was done following ANVISA validation guide after which red dry wine and white dry wine samples were analyzed. The proposed method was developed, having its functionalities attested and its analysis conditions enhanced, using 22% of ACN and 6 minutes in sample loading. The method was validated, demonstrating proper sensitivity, with detection and quantification limits equal to 0.02 and 0.05 µg L-1, respectively. Linearity and precision were evaluated, exhibiting correlation coefficient equal to 0,996 e RSD under 6%, respectively. The method was accurate in medium and high concentration levels and maximum recovery was 73% in high concentration level. Brazilian and foreign red dry wine and white dry wine samples were analyzed and OTA was not detected in any of them. However, OTA may be present in samples analyzed in lower concentrations than that ones determined by detection and quantification limits, not being a potential danger to human health.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2018-08-20T12:37:22Z No. of bitstreams: 1 2010 - Raquel Duarte da Costa Cunha Bandeira.pdf: 1478855 bytes, checksum: f7637b4df6dd802e4e0003f4fb48d8cd (MD5) / Made available in DSpace on 2018-08-20T12:37:22Z (GMT). No. of bitstreams: 1 2010 - Raquel Duarte da Costa Cunha Bandeira.pdf: 1478855 bytes, checksum: f7637b4df6dd802e4e0003f4fb48d8cd (MD5) Previous issue date: 2010-09-24 / Coffee is an extremely complex food matrix and has an important role in the world?s economy, especially in producing and exporting countries like Brazil. However this product may suffer from technical barriers imposed for exportation because of the possible presence of ochratoxin A, which is nefrotoxic and carcinogenic mycotoxin found in many foods including coffee. The aim of this study was to implement chemical metrology concepts in the development and validation of Liquid Chromatography with Mass Spectrometry in tandem (CLAE-EM/EM) method for identification and quantification of ochratoxin A in green and roasted coffee estimating uncertainty of measurement according to directive 2002/657/EC and Inmetro guidelines (DOC-CGCRE-2010). The extraction method was based on Pittet (1998) and chromatographic parameters were: flow rate of 0.3 mL/min, mobile phase 80:20 water trifluoracetic acid 0.05 %: methanol trifluoracetic acid 0.05 %, injection volume of 50 PL, injection mode Full loop, isocratic mode. The column was Synergi Hydro C18. The mass spectrometry parameters were optimized and transitions selected based on the colision energies monitored were m/z 404 >358 (-10.5 V) and m/z 404 >239 (-20.5 V). From the validation procedure, methods were considered seletive. The evaluation and verification of matrix effect was performed by comparing variances and averages using F and t test. Value of Fcalculated for green coffee (25.2152) and roasted coffee (104.0353), were higher than Ftable (4.0426). Value of t calculated for green (5.0214) and roasted coffee (10.1997) were higher than ttable (2.0106). Both methods were considered linear in the working range of calibration curve with linear correlation coefficients (r) of 0.98188 and 0.91754 for green and roasted coffee, respectively.The quantification and detection limits were 1.2 Pg/kg and 3.0 Pg/kg; 0.36 Pg/kg and 1.0 Pg/kg, for green and roasted coffee respectively. The average recoveries, RSDr and RSDR were in range of 90.45 ? 108.81 %, 5.39 ? 9.94 % and 2.20 ? 14.34 % for green coffee and 89.02 ? 108.85 %, 2.43 ? 13.73 % and 12.57 ? 17.84 % for roasted coffee. All results obtained were considered within acceptable levels according to literature. Measurement value and expanded uncertainties (U) for ochratoxin A were mass fraction w = (11.50 ? 1.11) and w = (4.63 ? 0.63) for green coffee and roasted coffee. Both methods developed and validated using a high sensitivity technique, that allowed detection, confirmation and quantification of ochratoxin A in green and roasted coffee with a estimated uncertainty of measurement, and in the future these methods can be used to help overcome possible technical barriers imposed for exportation of Brazilian coffee. / O caf? constitui uma matriz extremamente complexa e tem importante papel na economia mundial, especialmente nos pa?ses produtores e exportadores como o Brasil. No entanto tem sido alvo de barreiras t?cnicas devido a uma subst?ncia denominada ocratoxina A, micotoxina potencialmente nefrot?xica e nefrocarcinog?nica encontrada em muitos alimentos inclusive o caf?. O presente trabalho tem como objetivo implantar os conceitos da metrologia qu?mica no desenvolvimento, e valida??o do m?todo para identifica??o e quantifica??o de ocratoxina A no caf? verde e caf? torrado estimando a incerteza da medi??o e utilizando a t?cnica de Cromatografia L?quida acoplada a Espectrometria de Massas em s?rie (CLAE-EM/EM) seguindo os crit?rios da diretiva EC-657/2002 e o documento orientativo do Inmetro (DOCCGCRE- 2010). A metodologia de extra??o baseou-se em Pittet (1998) e os par?metros cromatogr?ficos foram: fluxo de 0,3 mL/min, fase m?vel 80:20 ?gua ?cido trifluoroac?tico 0,05%: metanol ?cido trifluoroac?tico 0,05 %, volume de inje??o de 50 PL, com o modo de inje??o Full loop e sistema de elui??o isocr?tico. A coluna utilizada foi Synergi Hydro C18. As condi??es do espectr?metro de massas foram otimizadas e a transi??o selecionada de acordo com suas energias de colis?o foram m/z 404 >358 (-10,5 V) e m/z 404 >239 (-20,5 V). A partir da valida??o os m?todos propostos foram considerados seletivos, a avalia??o e comprova??o do efeito matriz foi realizada atrav?s da compara??o das vari?ncias e das m?dias atrav?s do teste F e teste t. O Fcalculado para o m?todo caf? verde (25,2152) e caf? torrado (104,0353), apresentaram valores maiores que o Ftabelado (4,0426). O tcalculado para o caf? verde (5,0214) e torrado (10,1997) apresentaram valores superiores ao ttabelado (2,0106). Os m?todos foram considerados lineares em toda a faixa de trabalho da curva de calibra??o com os coeficientes de determina??o linear (r) de 0,98188 e 0,91754 para matriz caf? verde e caf? torrado, respectivamente. O limite de quantifica??o e detec??o para os m?todos propostos foram de 1,2 Pg/kg e 3,0 Pg/kg para caf? verde e 0,36 Pg/kg e 1,0 Pg/kg para caf? torrado. Os valores das recupera??es m?dias, DPRr e DPRR variaram na faixa de 90,45 - 108,81 %, 5,39 - 9,94 % e 2,20 - 14,34 % para caf? verde; e de 89,02 - 108,85 %, de 2,43 - 13,73 % e 12,57 - 17,84 %, para caf? torrado. Todos os resultados obtidos encontram-se dentro dos limites comumente aceit?veis na literatura. Todos os resultados de medi??o e as incertezas expandidas (U) para ocratoxina A foram as fra??es m?ssicas W = (11,50 ? 1,11) Pg/kg e W = (4,63 ? 0,63) Pg/kg para caf? verde e caf? torrado, respectivamente. Os m?todos desenvolvidos e validados utilizaram t?cnica de elevada sensibilidade, permitindo a detec??o, confirma??o e a quantifica??o de ocratoxina A no caf? verde e caf? torrado com c?lculo da incerteza, podendo auxiliar futuramente na supera??o das barreiras t?cnicas para exporta??o do caf? brasileiro.

As micotoxinas são compostos produzidos por fungos, sendo potencialmente perigosos à saúde humana e animal. A Ocratoxina A (OTA) é uma das micotoxinas mais amplamente estudadas, sendo encontrada em várias matrizes alimentícias. A concentração dessa micotoxina nos alimentos é geralmente muito baixa (da ordem de ng g-1), sendo, portanto, necessário o emprego de técnicas de preparo de amostras que realizem a purificação e pré-concentração no analito. Os métodos de separação e detecção empregados na análise de OTA também devem oferecer sensibilidade adequada para a quantificação do analito. Logo, os objetivos do trabalho foram desenvolver uma metodologia utilizando a técnica in-tube SPME no modo de extração única (flow through extraction) com partículas de C18 como fase extratora e separação e detecção por HPLC-MS/MS. Além disso, otimizar e validar a metodologia proposta e detectar e quantificar a OTA em amostras de vinho. Para isso, a técnica in-tube SPME foi configurada e um tubo de PEEK foi empacotado com partículas de C18, o qual foi utilizado na extração. A otimização do método foi realizada utilizando-se um planejamento experimental composto central 22 + 3 pontos centrais, tendo como fatores a porcentagem de ACN e o tempo durante o carregamento da amostra. A validação da metodologia empregada foi realizada conforme o guia de validação da ANVISA e, posteriormente, amostras de vinho tinto seco e vinho branco seco foram analisadas. O método proposto foi desenvolvido, tendo sua funcionalidade atestada e suas condições de análise melhoradas, sendo utilizado 22 % de ACN e 6 minutos no carregamento da amostra. O método foi validado, mostrando sensibilidade adequada, com limites de detecção e quantificação iguais à 0,02 e 0,05 µg L-1, respectivamente. A linearidade e precisão da metodologia foram avaliadas, apresentando coeficiente de correlação igual a 0,996 e DPR menor que 6%, respectivamente. O método mostrou-se exato nos níveis de concentração médio e alto e a recuperação máxima obtida para o nível alto foi próximo 73%. Amostras brasileiras e estrangeiras de vinho tinto seco e vinho tinto seco branco foram analisadas e a OTA não foi detectada em nenhuma delas. Entretanto, a OTA pode essa estar presente nas amostras analisadas em concentrações mais baixas que as determinadas pelos limites de detecção e quantificação, não sendo potencialmente perigosos à saúde. / Mycotoxins are compounds produced by fungus, being a potential danger to human and animal health. Ochratoxin A (OTA) is a mycotoxin widely studied, being found in a varied of food matrices. OTA concentration in food is generally low (the order of ng g-1), being, therefore, necessary the use of sample preparation techniques to perform the analyte purification and preconcentration in the sample. The separation and detection methods used in OTA analysis also should offer proper sensibility in order to quantify the analyte adequately. In short, the goals of the present work were to develop a methodology using in-tube SPME in flow through extraction mode with C18 particles as extraction phase and separation and detection by HPLC-MS/MS. Further, optimize and validate the proposed methodology and detect and quantify OTA in wine samples. For this, in-tube SPME was configured and a PEEK tube was packed with C18 particles, which was used in the extraction step. The method optimization was achieved using a central composite 22 + 3 central points experimental design, having as factors the percentage of ACN and time during sample loading step. Validation method was done following ANVISA validation guide after which red dry wine and white dry wine samples were analyzed. The proposed method was developed, having its functionalities attested and its analysis conditions enhanced, using 22% of ACN and 6 minutes in sample loading. The method was validated, demonstrating proper sensitivity, with detection and quantification limits equal to 0.02 and 0.05 µg L-1, respectively. Linearity and precision were evaluated, exhibiting correlation coefficient equal to 0,996 e RSD under 6%, respectively. The method was accurate in medium and high concentration levels and maximum recovery was 73% in high concentration level. Brazilian and foreign red dry wine and white dry wine samples were analyzed and OTA was not detected in any of them. However, OTA may be present in samples analyzed in lower concentrations than that ones determined by detection and quantification limits, not being a potential danger to human health.

The ochratoxin A (OTA) is a mycotoxin with nephrotoxic, nephrocarcinogenic, teratogenic and immunossupresive properties that can be found in grape juice and wine, as well as other food groups, like cereals and oil seeds. Its occurrence in grapes and afterwards in musts and wines is due mainly to the phytosanitary conditions of grapes, besides their variety, the degree of berry maturity, physical damage, viticulture practices and climatic conditions; once the ochratoxin A is produced by strains of Penicillium and Aspergillus species. The objective of this study was to determine the relationship between the phytosanitary conditions of grape and the level of ochratoxin A in white vitis vinifera musts and wines. The grapes were collected at Cooperativa Vinícola Aurora Ltda., localized in Pinto Bandeira, a district of Bento Gonçalves, RS from three varieties of white vitis vinifera (Gewürztraminer, Chardonnay and Sauvignon Blanc). The grapes were collected four (4)days apart from the harvest day stablished by the vineyard (day zero) up to the eighth (8) (to Gewürztraminer and Sauvignon Blanc wines) and fifteenth (15) days (Chardonnay). The maturation criteria were the º Brix and total acidity determinations. The collected amount of grape in each sample was approximately 6 Kg and three repetitions of each sampling (to each grape variety), were made. The plant s selection was randomized, by the collection of a small amount of bunches in each sample. The grape microvinification was made after the harvest. In the musts was realized the determination of pH, soluble solids (º Brix) and total acidity. In all the wine samples these physic-chemical parameters were analyzed: alcohol content, density, pH, total acidity, volatile acidity; total sugar and reduced dry stratum. The determination of ochartoxin A in musts and wines was done by High-Performance Liquid Chromatography (HPLC) with fluorescence detection accordingly to the of Office International de La Vigne et Du Vin (OIV) (2001), with the use of immunoaffinity columns. Statistical analysis was performed using the program SAS for Windows 2000, version 6.11. Even presenting different conditions of sanity, neither the musts nor the wines had detectable levels of ochratoxin A. The physic-chemical analysis was in the range accepted by the legislation of wines production for beverage. / A Ocratoxina A (OTA) é uma micotoxina que possui propriedades nefrotóxicas, nefrocarcinogênicas, teratogênicas e imunossupressora que pode ser encontrada no vinho e no suco de uva, bem como em outros grupos de alimentos, como cereais e oleaginosas. A sua ocorrência nas uvas e posteriormente nos mostos e nos vinhos, deve-se principalmente às condições fitossanitárias das uvas, além da sua variedade, grau de maturação, danos físicos do grão, práticas de viticultura e condições climáticas, uma vez que a Ocratoxina A é produzida por espécies de fungos dos gêneros Penicillium e Aspergillus. O objetivo principal do presente trabalho foi determinar a relação entre as condições fitossanitárias da uva com o teor de Ocratoxina A dos mostos e vinhos de variedades viníferas brancas. As uvas foram coletadas na Cooperativa Vinícola Aurora Ltda., na localidade de Pinto Bandeira, do município de Bento Gonçalves, RS, a partir de três variedades de uva viníferas brancas, Gewürztraminer, Chardonnay e Sauvignon Blanc. As uvas foram coletadas num intervalo de aproximadamente quatro em quatro (4) dias, após a data de colheita estabelecida pela vinícola (que foi o dia zero) até o oitavo (8) dia após esta data, para as variedades Gewürztraminer e Sauvignon Blanc e até o décimo quinto dia (15) para a variedade Chardonnay. O critério de maturação foi a determinação do grau Brix e da acidez total. O volume coletado em cada amostragem foi de aproximadamente 6 Kg de uva sendo que foram feitas três repetições de cada amostragem, para cada variedade de uva. A escolha das plantas foi realizada de forma aleatória, coletando um pequeno número de cachos das plantas em cada amostragem. Posteriormente à colheita, foram realizadas as microvinificações destas uvas. Nos mostos foram realizadas as determinações de pH, sólidos solúveis (º Brix) e acidez total. Em todas as amostras dos vinhos foram analisados parâmetros físico-químicos como: teor alcoólico; densidade; pH; acidez total e volátil; açúcares totais e extrato seco reduzido. Ocratoxina A foi determinada nos mostos e vinhos através de Cromatografia Líquida de Alta Eficiência com detecção por fluorescência, segundo a técnica descrita pela Office International de La Vigne et Du Vin (OIV) (2001), com o uso de colunas de imunoafinidade. Os resultados das análises dos mostos e vinhos foram avaliados estatisticamente através do programa computacional SAS for Windows 2000, versão 6.11. Tanto as amostras dos mostos como dos vinhos analisados não apresentaram teores detectáveis de ocratoxina A, mesmo sendo utilizadas uvas de diferentes condições de sanidade. Já em relação às análises físico-químicas dos mostos e dos vinhos, todas se encontraram dentro dos limites exigidos pela legislação para a produção de vinhos ideais para consumo.

Un appareil de mesure de la fluorescence, à faible coût et portable a été développé pour quantifier les concentrations d’Ochratoxine A (OTA) dans des échantillons réels. Le système est basé sur l’excitation par une UV-LED à 365 nm et un photo détecteur contrôlé par une interface dans LabVIEW. Aussi, une image capteur, CMOS, contrôlée par une interface conçue dans MATLAB. L’OTA est une molécule naturellement fluorescente. Après excitation par une UV-, l’image de la fluorescence émise est captée par une caméra et traitée en vue de la mesure de la concentration de l’OTA. Le système d’analyse a été basé sur les 3 composants rouge, vert et bleu (RGB, selon l'acronyme anglais). La gamme est linéaire entre de 2-40 µg/L. L’extraction de l’OTA est réalisée par des colonnes d'immuno affinité (IAC, selon l'acronymeanglais) et les colonnes à empreinte moléculaire (MIP, selon l'acronyme anglais) pour les échantillons de cacao, de la bière et du vin. Les résultats obtenus ont été validés par la méthode chromatographique (HPLC). L'appareil conçu est facile à utiliser, économique et portable. En outre, l'utilisation des nouvelles technologies a été inclus tels que l'emploi du smartphone pour détecter l'OTA et la création d'un APP. Des données d'image de fluorescence provenant de la caméra du smartphone et sont analysées par un ordinateur personnel et présentés dans les composantes RGB, où l'image est envoyée à l'ordinateur par WIFI et le téléphone intelligent est utilisé comme source d'énergie trop. Enfin, une APP pour le système Android a été créé pour capturer l'image et fournit les valeurs RGB. Enfin l'utilisation du traitement de l'image a été utilisée pour quantifier l'OTA dans les échantillons réels sans colonnes IAC ou MIP, employée pour extraire la mycotoxine. L’analyse a été réalisée par des techniques colorimétriques et d’analyse de la couleur. / A portable and low cost fluorescence set-up to quantify the concentrations of Ochratoxin A(OTA) in real samples was developed. The detection through the device consist of anultraviolet light at 365 nm and an photo detector or a CMOS sensor controlled by anexecutable interface designed in LabVIEW or MATLAB. It has been reported that OTA is naturally fluorescent, so it allows the user to get a UV LED to excite the sample, get a value involtage when a photodetector is employed or a photograph of the OTA under excitationconditions, and process that image in order to predict the concentrations of the sample. Tocapture and process the image, in an automatically manner, the system was completely basedon the Red, Green and Blue (RGB) components. The linearity for OTA obtained in the rangeof concentrations corresponds to 2-40 µg/L. Immunoaffinity columns (IAC) and molecularimprinted polymer columns (MIP) were used with cocoa, beer and wine samples. Theobtained results were cross-validated using chromatographic method such as HPLC and theFluoroskan equipment. The developed setup is easy to use, economical and portable. Besides,the use of new tendencies was included such as employ the smartphone to detect OTA and thecreation of an APP. Fluorescence image data from the smartphone camera are analyzed by apersonal computer and presented in RGB components, where the image is sent to thecomputer by WIFI and the smartphone is used as a power source too. Finally, an APP forandroid system was created to capture the image and provides the RGB values. At the end, theuse of image processing to quantify the OTA in real samples without incorporated IAC or MIPcolumns to extract the mycotoxin from a complex solution, employing colorimetric techniquesand color analysis.