Suchergebnisse

Mycotoxins attract worldwide attention because of the significant economic losses associated with their impact on human health, animal production and both domestic and international trade. Those mycotoxins that are currently considered to be worldwide importance are aflatoxins, trichothecenes, zearalenone, fumonisins, ochratoxin A, patulin (Coker, 2000). The knowledge that mycotoxins can have serious effects on humans and animals has led many countries to establish regulations on mycotoxins in food and feed in the last decades to safeguard the health of humans, as well as the economical interests of producers and traders. Setting mycotoxin regulations is a complex activity which involves many factors and interested parties. In 1995, 23 percent of the world's inhabitants were living in a region where no known mycotoxin regulations were in force. This percentage had decreased to 13 percent in 2003, due to a slight increase in coverage in Latin America and Europe, and more significant increases in Africa and Asia/Oceania.The major Latin American agricultural crops (maize, wheat, coffee, cotton, soybeans, barley, sunflower, groundnuts and tree nuts, cocoa and dairy products) are highly susceptible to fungal contamination and mycotoxin production (Pineiro, 2004). Nineteen countries, accounting for 91 percent of the population of the region, were known to have specific mycotoxin regulations. Uruguay has the most detailed regulations, including limits for ergot alkaloids in feeds, which is rather unique in the mycotoxin regulatory world. The same for deoxynivalenol in wheat products and barley products.MERCOSUR consists of Argentina, Brazil, Paraguay and Uruguay. These countries apply common limits for total aflatoxins in peanuts, maize and products thereof, and for aflatoxin M1 in fluid and powdered milk. The MERCOSUR regulations for mycotoxins also include official methods of sampling and analysis. In Europe, approximately 99 percent of the continent's population, were known to have specific mycotoxin regulations in 2003.Compared to other regions of the world, Europe has the most extensive and detailed regulations for mycotoxins in food. It is of interest to note that many of the EU candidate member countries have mycotoxin regulations, which are often more detailed than those currently in force in the EU. Comparing the situation in 1995 and 2003, it appears that in 2003 more mycotoxins are regulated in more commodities and products, whereas tolerance limits generally remain the same or tend to decrease.Whereas harmonized tolerance limits would be beneficial from the point of view of trade, this would not necessarily be the case from the point of view of (equal) human health protection around the world. Risks associated with mycotoxins depend on both hazard and exposure. The hazard of mycotoxins to individuals is probably more or less the same all over the world .Exposure is not the same because of differences in levels of contamination and dietary habits in various parts of the world. National governments or regional communities should encourage and fund activities that contribute to reliable exposure assessment of mycotoxins in their regions.(FAO Food and Nutrition paper 81)

Mycotoxins contaminants, one of the most serious global challenges, have been attracted more and more attention from International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) and scientists in food safety sciences. Many countries and organizations have established the maximum contamination level of the main mycotoxins at very low values. Traditional analytic strategies are mainly based on instrumental quantitative method and immunoassays approaches. Aptamer, a novel molecules recognition element like or even superior to antibodies, has attracted more and more attentions for scientists. Therefore, specific aptamers could be employed to construct biosensors for the detection of mycotoxins. The objective of this thesis is to develop novel aptamer-based biosensors for sensitive determination of trace levels of mycotoxins and to provide a promising application of these aptasensors for more hazard factors in food safety sciences. The main contents and results are as follows: (1) Aptamer-based biosensor for detection of mycotoxins Mycotoxins are a large types of secondary metabolites appeared by fungi, they pose a great hazard and toxic reactions to human and animals. A majority of countries and regulators, such as European Union, have established series of requirements and set the maximum tolerated levels. The development of high sensitive and specific analytical platform for mycotoxins is much in demand to address new challenges for food safety in worldwide. Due to the superiority of simple, rapid, and low-cost characteristics, aptamer-based biosensors are successfully developed for the detection of various mycotoxins with high sensitivity and selectivity compared with traditional instrumental methods and immunological approaches. In this article, we discuss and analyze the development of aptasensors for mycotoxins determination in food and agricultural products during the last eleven years and cover the literatures from the first report in 2008 until today. In addition, challenges and future trends for the selection of aptamers towards various mycotoxins and aptasensors for multi-mycotoxins analysis are summarized. Given the promising development and potential application of aptasensors, the future researches will witness the great practicability of aptamer-based biosensor for food safety field. (2) A qPCR aptasensor for sensitive detection of aflatoxin M1 Aflatoxin M1 (AFM1), one of the most toxic mycotoxins, imposes serious health hazards. AFM1 had previously been classified as a group 2B carcinogen (IARC, 1993) and has been classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) (IARC, 2002). Determination of AFM1 thus plays an important role for quality control of food safety. In this work, a sensitive and reliable aptasensor was developed for the detection of AFM1. The immobilization of aptamer through a strong interaction with biotin–streptavidin was used as a molecular recognition element, and its complementary ssDNA was employed as the template for a real-time quantitative polymerase chain reaction (RT-qPCR) amplification. Under optimized assay conditions, a linear relationship (ranging from 1.0×10-4 to 1.0 µg L-1) was achieved with a limit of detection (LOD) down to 0.03 ng L-1. In addition, the aptasensor developed here exhibits high selectivity for AFM1 over other mycotoxins and small effects from cross-reaction with structural analogs. The method proposed here has been successfully applied to quantitative determination of AFM1 in infant rice cereal and infant milk powder samples. Results demonstrated that the current approach is potentially useful for food safety analysis, and it could be extended to a large number of targets. (3) A novel graphene oxide-based aptasensor for amplified fluorescent detection of aflatoxin M1 in milk powder In this paper, a rapid and sensitive fluorescent aptasensor for the detection of aflatoxin M1 (AFM1) in milk powder has been developed. Graphene oxide (GO) was employed to quench the fluorescence of carboxyfluorescein-labelled aptamer and protect the aptamer from nuclease cleavage. Upon the addition of AFM1, a formation of AFM1/aptamer complex resulted in the aptamer detached from the surface of GO, then the aptamer was cleaved by DNase I and the target AFM1 was released for a new cycle, which led to a great signal amplification and high sensitivity. Under optimized conditions, the GO-based detection of the aptasensor exhibited a linear response to AFM1 in a dynamic range from 0.2 to 10 μg/kg, with a limit of detection (LOD) of 0.05 μg/kg. Moreover, the developed aptasensor showed a high specificity towards AFM1 without interference from other mycotoxins. In addition, the technique has been successfully applied for detection of AFM1 in infant milk powder samples. This aptasensor proposed here offers a promising technology for food safety and can be extended to various targets. (4) Graphene oxide driven fluorescent aptasensor for the detection of fumonisin B1 Fumonisin B1 (FB1), one of the most toxic mycotoxins, has been designated as possible 2B group carcinogen by the International Agency for Research on Cancer (IARC) in 2002. Therefore, simple, sensitive and specific approaches for the detection of FB1 are much in demand. In this study, a novel aptasensor was introduced for FB1 analysis based on graphene oxide (GO) and DNase I signal amplification. GO was adopted as a fluorescence quencher against ROX-modified aptamer and a protectant for the aptamer from cleavaging by DNase I for subsequent target cycling and signal amplification detection. This proposed sensing strategy exhibited a good linearity for FB1 determination in the dynamic range from 0.5 to 20 ng mL-1 with a good correlation of R2 = 0.995. Its detection of limit was established at 0.15 ng mL-1 (S/N = 3). The specific analysis indicated that this aptasensor was selective for FB1 other than other mycotoxins. In addition, the practical application in real samples of this aptasensor for the detection of FB1 was investigated. The sensing platform proposed here was useful for a potential application in the field of food safety for mycotoxins analysis. (5) Articles highlights and future perspective By using the novel aptamer-based biosensors, we developed several approaches for the detection of the most toxic mycotoxins for food safety. In addition, these sensing strategies could be applied for more hazard factors determination by simple replacement of the specific aptamers. More importantly, though the practical applicability, feasibility, and accuracy of these proposed aptasensors were investigated and evaluated through the analysis of the spiked samples experiments, the future's researches were needed for a validation of these aptasensors with real contaminated samples to determine the performances such as limit of detection and quantification, precision, trueness, accuracy, etc. Through the method validation, these aptasensors will be widely used for the detection of mycotoxins. In addition, future direction will focus on the simplification of analytic principle and devices and the combination of novel aptamers with new materials and techniques to improve the analytical performance and market practicality of aptasensors.

Dissertação de Mestrado em Segurança Alimentar apresentada à Faculdade de Farmácia ; The consumption of pistachios (Pistacia vera L.) has been increasing, given its important benefit in human health. In addition to an excellent nutritional source, it has associated chemical hazards, such as mycotoxins, resulting of fungal contamination and its secondary metabolism. Pistachios are one of nuts with higher mycotoxin's contamination worldwide, especially Aflatoxin B1 with a hepatotoxic effect and classified as proven carcinogenic to humans by the International Agency for Research on Cancer (IARC). More mycotoxins as ochratoxin A (OTA), fumonisins (FBs), zearalenone (ZEA) and trichothecenes (T2, HT2 and DON) and emerging mycotoxins have been concerned in nuts. This study developed a Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method followed by Ultra-High Performance Liquid Chromatography combined with Time-of-Flight Mass Spectrometry (UHPLC–ToF-MS) for the determination of multi-mycotoxins in pistachios.Different approaches in dispersive solid phase extraction (d-SPE) as clean-up for high-lipid matrix were to evaluate. For this, classic sorbents, like C18 (octadecyl modified silica) and PSA (primary secondary amine) and new classes of sorbents, namely EMR-Lipid (enhanced matrix removal-lipid) and Z-Sep (modified silica gel with zirconium oxide) are used. Method with 100 mg Z-Sep sorbent provided the best analytical performance, with good recovery (79 to 120%), repeatability (RSDr<10%) and precision inter-day (RSDR<10%) in agreement with criteria established by Commission Regulation EC No. 401/2006 for mycotoxins analysis. The method was validated for aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), zearalenone (ZEA), toxin T2 (T2) and toxin HT-2 (HT2). The LODs for AFs ranged from 0.125 to 0.25 ug/Kg, which are lower than the maximum levels in nuts regulated by the EU. The method was applied to 16 real pistachio samples and 6 of these presented one mycotoxin (AFB1, HT2 or FB1) but at low concentrations. The concentration of AFB1 was lower than the maximum permitted level established by the EU legislation. Also, AFB2 and FB1 are detected in pistachio shells. In this line, pistachios samples selected in the present study and available in the Portuguese market are safe for human consumption concerning mycotoxins content. ; O consumo de pistácios (Pistacia vera L.) tem vindo a aumentar devido ao seu reconhecido benefício na saúde humana. Apesar de constituírem uma excelente fonte nutricional, os pistácios têm riscos químicos associados, como as micotoxinas, resultantes da contaminação de fungos e do seu metabolismo secundário. Os pistácios são um dos frutos secos com maior contaminação por micotoxinas em todo o mundo, especialmente por aflatoxinas. As aflatoxinas são as mais tóxicas para os seres humanos, particularmente a aflatoxina B1 com efeito hepatotóxico e classificada como comprovadamente carcinogénica para o homem pela Agência Internacional para a Investigação do Cancro (IARC). Outras micotoxinas são relevantes como ocratoxina A (OTA), fumonisinas (FBs), zearalenone (ZEA) e trichotecenes (T2, HT2 e DON) e mais recentemente, as micotoxinas emergentes.Este estudo desenvolveu um método rápido, fácil, barato, eficaz, robusto e seguro (QuEChERS) seguido de Cromatografia Líquida de Ultra Resolução combinada com Espectrometria de Massa de Tempo de Voo (UHPLC-ToF-MS) para a determinação das micotoxinas em pistácios. Diferentes abordagens no clean-up pela técnica de extração de Fase Sólida Dispersiva (d-SPE) foram avaliadas. Para isso, foram utilizados adsorventes clássicos, como C18 (octadecil sílica) e PSA (amina secundária primária) e novas classes de adsorventes, nomeadamente, EMR-Lipid (enhanced matrix removal-lipid) e Z-Sep (gel de sílica modificada com óxido de zircónio). O método com 100 mg de Z-Sep proporcionou o melhor desempenho analítico, com uma boa recuperação (79 a 120%), boa repetibilidade (RSDr<10%) e a boa precisão inter-dia (RSDR<10%) de acordo com critérios estabelecidos pelo Regulamento N.º 401/2006 da Comissão Europeia para a análise das micotoxinas. O método foi validado para aflatoxinas (AFB1, AFB2, AFG1 e AFG2), ocratoxina A (OTA), zearalenona (ZEA), toxina T2 (T2) e toxina HT-2 (HT2). Os LODs para as AFs variaram entre 0,125 e 0,25 ug/Kg, concentrações inferiores aos níveis máximos para frutos secos regulados pela UE. O método foi aplicado a 16 amostras de pistácios e em 6 destas foi determinada uma micotoxina (AFB1, HT2 ou FB1), mas em baixas concentrações. A concentração de AFB1 foi inferior ao valor máximo permitido de acordo com a legislação da União Europeia. Além disso, AFB2 e FB1 foram detetadas nas cascas de pistácios. Assim, os pistácios selecionados para o presente estudo e disponíveis no mercado português são considerados seguros para o consumo humano no que diz respeito à presença de micotoxinas. ; FCT

Dissertação de Mestrado em Segurança Alimentar apresentada à Faculdade de Farmácia ; The consumption of pistachios (Pistacia vera L.) has been increasing, given its important benefit in human health. In addition to an excellent nutritional source, it has associated chemical hazards, such as mycotoxins, resulting of fungal contamination and its secondary metabolism. Pistachios are one of nuts with higher mycotoxin's contamination worldwide, especially Aflatoxin B1 with a hepatotoxic effect and classified as proven carcinogenic to humans by the International Agency for Research on Cancer (IARC). More mycotoxins as ochratoxin A (OTA), fumonisins (FBs), zearalenone (ZEA) and trichothecenes (T2, HT2 and DON) and emerging mycotoxins have been concerned in nuts. This study developed a Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method followed by Ultra-High Performance Liquid Chromatography combined with Time-of-Flight Mass Spectrometry (UHPLC–ToF-MS) for the determination of multi-mycotoxins in pistachios.Different approaches in dispersive solid phase extraction (d-SPE) as clean-up for high-lipid matrix were to evaluate. For this, classic sorbents, like C18 (octadecyl modified silica) and PSA (primary secondary amine) and new classes of sorbents, namely EMR-Lipid (enhanced matrix removal-lipid) and Z-Sep (modified silica gel with zirconium oxide) are used. Method with 100 mg Z-Sep sorbent provided the best analytical performance, with good recovery (79 to 120%), repeatability (RSDr<10%) and precision inter-day (RSDR<10%) in agreement with criteria established by Commission Regulation EC No. 401/2006 for mycotoxins analysis. The method was validated for aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), zearalenone (ZEA), toxin T2 (T2) and toxin HT-2 (HT2). The LODs for AFs ranged from 0.125 to 0.25 ug/Kg, which are lower than the maximum levels in nuts regulated by the EU. The method was applied to 16 real pistachio samples and 6 of these presented one mycotoxin (AFB1, HT2 or FB1) but at low concentrations. The concentration of AFB1 was lower than the maximum permitted level established by the EU legislation. Also, AFB2 and FB1 are detected in pistachio shells. In this line, pistachios samples selected in the present study and available in the Portuguese market are safe for human consumption concerning mycotoxins content. ; O consumo de pistácios (Pistacia vera L.) tem vindo a aumentar devido ao seu reconhecido benefício na saúde humana. Apesar de constituírem uma excelente fonte nutricional, os pistácios têm riscos químicos associados, como as micotoxinas, resultantes da contaminação de fungos e do seu metabolismo secundário. Os pistácios são um dos frutos secos com maior contaminação por micotoxinas em todo o mundo, especialmente por aflatoxinas. As aflatoxinas são as mais tóxicas para os seres humanos, particularmente a aflatoxina B1 com efeito hepatotóxico e classificada como comprovadamente carcinogénica para o homem pela Agência Internacional para a Investigação do Cancro (IARC). Outras micotoxinas são relevantes como ocratoxina A (OTA), fumonisinas (FBs), zearalenone (ZEA) e trichotecenes (T2, HT2 e DON) e mais recentemente, as micotoxinas emergentes.Este estudo desenvolveu um método rápido, fácil, barato, eficaz, robusto e seguro (QuEChERS) seguido de Cromatografia Líquida de Ultra Resolução combinada com Espectrometria de Massa de Tempo de Voo (UHPLC-ToF-MS) para a determinação das micotoxinas em pistácios. Diferentes abordagens no clean-up pela técnica de extração de Fase Sólida Dispersiva (d-SPE) foram avaliadas. Para isso, foram utilizados adsorventes clássicos, como C18 (octadecil sílica) e PSA (amina secundária primária) e novas classes de adsorventes, nomeadamente, EMR-Lipid (enhanced matrix removal-lipid) e Z-Sep (gel de sílica modificada com óxido de zircónio). O método com 100 mg de Z-Sep proporcionou o melhor desempenho analítico, com uma boa recuperação (79 a 120%), boa repetibilidade (RSDr<10%) e a boa precisão inter-dia (RSDR<10%) de acordo com critérios estabelecidos pelo Regulamento N.º 401/2006 da Comissão Europeia para a análise das micotoxinas. O método foi validado para aflatoxinas (AFB1, AFB2, AFG1 e AFG2), ocratoxina A (OTA), zearalenona (ZEA), toxina T2 (T2) e toxina HT-2 (HT2). Os LODs para as AFs variaram entre 0,125 e 0,25 ug/Kg, concentrações inferiores aos níveis máximos para frutos secos regulados pela UE. O método foi aplicado a 16 amostras de pistácios e em 6 destas foi determinada uma micotoxina (AFB1, HT2 ou FB1), mas em baixas concentrações. A concentração de AFB1 foi inferior ao valor máximo permitido de acordo com a legislação da União Europeia. Além disso, AFB2 e FB1 foram detetadas nas cascas de pistácios. Assim, os pistácios selecionados para o presente estudo e disponíveis no mercado português são considerados seguros para o consumo humano no que diz respeito à presença de micotoxinas. ; FCT

Disease outbreaks due to the consumption of contaminated food and feedstuff are a recurring problem worldwide. The major factor contributing to contamination are microorganisms, especially fungi, which produce low-molecular-weight compounds as secondary metabolites, with confirmed toxic properties referred to as mycotoxins. Mycotoxins are toxic secondary metabolites produced by fungi that invade crops at the field level and may grow on foods during storage under favorable conditions. The toxigenic fungi of Aspergillus, Penicillium, Fusarium, Alternari and Clavicepshave genera are of the greatest consequence to food safety. Mycotoxins, of over 400 that are known, which have the most food safety, nutritive, ecologic and economic significance include the aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins, tremorgenic mycotoxinsand ergotalcaloides. Some molds are capable of producing more than one mycotoxin and some mycotoxins are produced by more than one fungal species. Often more than one mycotoxin is found on a contaminated substrate. Factors influencing the presence of mycotoxins in foods or feeds include environmental conditions related to storage that can be controlled. Other extrinsic factors such as climate or intrinsic factors such as fungal strain specificity, strain variation, and instability of toxigenic properties are more difficult to control. Exposure to mycotoxins is mostly by ingestion, but also occurs by the dermal and inhalation routes. The diseases caused by exposure to mycotoxins are known as mycotoxicoses. Mycotoxins have various acute and chronic effects on humans and animals (especially monogastrics) depending on species and susceptibility of an animal within a species. Ruminants, however, are generally more resistant to the adverse effects of mycotoxins. This is because the rumen microbiota is capable of degrading mycotoxins. The economic impact of mycotoxins include loss of human and animal life, increased health care and veterinary care costs, reduced livestock production, disposal of contaminated foods and feeds, and investment in research and applications to reduce severity of the mycotoxin problem. This review is meant to be informative not only for health-conscious consumers but also for experts in the field to pave the way for future research to fill the existing gaps in our knowledge in regard to mycotoxins and food safety. In this review, the focus is on the occurrence of various types of mycotoxins in food and feed associated with risks to humans and livestock, as well as legislation put forth by various authorities. Brief descriptions on recent developments in mycotoxin detection methodology and on presently practiced detoxification methods are also included. ; Oboljenja ljudi prouzrokovana kontaminiranom hranom predstavljaju jedan od najvećih problema sa kojim se suočava savremeno čovečanstvo. Glavni uzročnici kontaminacije su mikroorganizmi, naročito plesni, koje sintetišu jedinjenja male molekulske mase sa izrazitim toksičnim efektom na žive organizme. Mikotoksini su sekundarni metaboliti pretežno Aspergillus, Penicillium, Fusarium, Alternaria i Claviceps vrsta plesni, koje mogu kontaminirati hranu na polju (preharvest) i/ili tokom skladištenja (postharvest). Iako je do sada poznato preko 400 mikotoksina zbog svoje zastupljenosti i toksičnosti, afl atoksini (AFT), ohratoksin A (OTA), trihoteceni (TCT), zearalenon (ZEA), fumonizini (FB), tremorgeni mikotoksini i ergotalkaloidi, predstavljaju najveći medicinski, nutritivni, ekološki i ekonomski problem. Specifičnost mikotoksina u odnosu na druge toksine ogleda se u tome da pojedini rodovi plesni mogu da sintetišu nekoliko mikotoksina, kao i to da pojedini mikotoksini mogu biti proizvod sekundarnog metabolizma nekoliko rodova i vrsta plesni. S toga je kozastupljenost mikotoksina u kontaminiranoj hrani veoma česta pojava. Faktori koji utiču na kolonizaciju plesni i sintezu mikotoksina odnose se na faktore spoljašnje sredine (ekstrinsik) u koje spadaju skladišni uslovi i koji se mogu kontrolisati, dok ostale faktore spoljašnje sredine kao što su klimatske promene ili unutrašnje (intrinsik) faktore, u koje spadaju specifičnost i varijacije pojedinih vrsta plesni i nestabilnost toksigenih svojstava plesni, je veoma teško kontrolisati. Mikotoksini u organizam ljudi i životinja najčešće dospevaju putem kontaminirane hrane, ali su inhalacioni i dermalni put, takođe mogući. Oboljenja ljudi i životinja izazvana mikotoksinima se nazivaju mikotoksikoze. Mikotoksini izazivaju različite akutne i hronične biološke efekte u organizmu ljudi i životinja. Smatra se da su monogastrične životinje daleko osetljivije na dejstvo mikotoksina u odnosu na preživare. Ekonomski značaj mikotoksina odražava se kroz povećane troškove lečenja ljudi i životinja, smanjenje produktivnih rezultata životinja uključujući i uginuća, direktne i indirektne štete koje nastaju usled uklanjanja kontaminirane hrane, investiranje u istraživanja i primenu preventivnih mera u sprečavanju negativnog efekta prisustva mikotoksina u hrani na zdravlje ljudi i životinja. Ovaj rad ima za cilj da sagleda ne samo zdravlje ljudi, već i da bude informativan za stručnjake u ovoj oblasti kako bi se otklonile određene nejasanoće vezane za prisustvo ove vrste hemijskog hazarda biološkog porekla u lancu ishrane. Stoga je u ovom radu prikazana zastupljenost i toksičnost najznačajnijih mikotoksina i način donošenja zakonske regulative. Takođe, opisane su analitičke metode za dokazivanje mikotoksina i mere koje se preduzimaju u prevenciji i kontroli mikotoksina.

This thesis concerns with the development and the validation of analytical methods for the determination of the mycotoxins aflatoxin B1, zearalenone and patulin, which occur frequently in food and feed. The toxic syndromes produced by them when ingested are known as mycotoxicoses. One of the first reports in history of mycotoxicoses is ergotism, caused by the fungus Claviceps purpurea. Nowadays ergotism is of minor importance; however the problem of mycotoxicoses and long term sub-acute exposure has not faded. Therefore, regulations have been established in many countries, and reliable testing methodology is needed to implement and enforce the regulatory limits. So far, several hundred different mycotoxins have been discovered, exhibiting different structural diversity, with various chemical and physicochemical properties, but only a few present significant food safety challenges. Among these are aflatoxins and ochratoxin A (produced by Aspergillus sp.), fumonisins, trichothecenes such as T-2, HT-2 toxins, deoxynivalenol and zearalenone (produced by Fusarium sp.), patulin (produced by Penicillium sp.) and ergot alkaloids (produced by Claviceps sp.) the most frequent occurring mycotoxins with the highest potential to adverse effects in humans and animals. The work of this thesis can be clustered into three parts as follows: (I) Method comparison and collaborative trial for the determination of aflatoxin B1 in medicinal herbs. This study was initiated upon the request of the European Pharmacopoeia since the regulatory limits for aflatoxin B1 in medicinal herbs were discussed in that moment. The methodology used has been adopted from existing methods for the determination of aflatoxin B1 in food. The food is extracted with an organic solvent followed by immunoaffinity clean-up and reversed-phase high performance liquid chromatography with fluorescence detection. The aim was to select the most suitable method parameters in order to obtain a method that allows the precise determination of aflatoxin B1 in a variety of medicinal herbs. Therefore acetone-water and methanol-water were tested as extraction solvents. Further, the influence of different post-column derivatisation options with electrochemically generated bromine, photochemical reaction and chemical bromination was compared. In addition, two different calculation modes peak height versus peak area were investigated concerning the precision on the evaluation of the rather small peaks that are obtained for aflatoxin B1 at low contamination levels. The different method parameters were applied in the collaborative study to three matrices: senna pods, ginger root and devil's claw root. As a result, the method with all tested variations was found to be fit-for-purpose for the determination of aflatoxin B1 in medicinal herbs at levels of 1 µg/kg and above. It could be concluded that the tested derivatisation methods had no influence on the analytical result in a range of 1 - 3 µg/kg for aflatoxin B1 in medicinal herbs. This is an interesting conclusion as control laboratories often have a preference for one or the other derivatisation method depending on their experience with one or the other system and its availability. 'A second method was adopted by single-laboratory validation for the determination of aflatoxin B1 in tiger nuts. The interest on tiger nuts rose on the fact of recent entries in the Rapid Alert System for Food and Feed regarding contamination with aflatoxin B1 in tiger nuts. This system allows the European Commission, EU member states and other associated countries to share information and take immediate action when potentially dangerous food or feed is detected on the market or at the border. Additionally a small survey of aflatoxin B1 content in chufa, which is a tiger nuts based soft drink in Spain, was conducted with the adopted method. The detection limit and the quantification limit were 0.02 µg/kg and 0.06 µg/kg respectively. The mean recovery at a level of 2 µg/kg was 88 % (n = 6) and the coefficient of variation 9 %. (II) Development and validation of an analytical method for the determination of zearalenone in infant food as well as in animal feed. The main challenge was to allow the determination of zearalenone at rather low concentrations in infant food, while being also able to deal with complex and more challenging matrices such as compound animal feed, due to their abundant interferences compounds. Previous work performed the determination of zearalenone in cereal grains and at higher levels. The method was validated in an international interlaboratory trial in which laboratories from EU member states, China, Turkey and Uruguay participated. Method performance parameters for both baby food and animal feed were calculated based on results for spiked samples blind duplicates at two levels and based on results for naturally contaminated samples blind duplicates at three levels. Test portions of the samples were spiked at levels of 20 µg/kg and 30 µg/kg zearalenone in baby food and at levels of 100 µg/kg and 150 µg/kg zearalenone in animal feed. As a result the method showed acceptable within-laboratory and between-laboratory precision for each matrix, as required by European legislation. Therefore, the newly developed method allows the enforcement of EU legislative limits for zearalenone in foods for infants at 20 µg/kg. (III) Development and validation of an analytical method for the determination of patulin in juices and purees for infants. The main challenge was to stress the method to determine patulin reliably at levels of 10 µg/kg in products intended for infants and young children. Previously developed methods for patulin were either collaboratively tested at higher levels, indicating that the lower limit for reliable quantification of patulin in such products was around 25 µg/kg or higher, or no validation data except single-laboratory validation were available. Method development focussed on improved and simplified extraction and clean-up procedures. A single liquid-liquid extraction in the presence of sodium sulfate as water binding agent showed sufficient extraction recovery rates for patulin in combination with a solid-phase extraction method, which trapped interfering substances and allowed the purification of patulin extracts without any pre-conditioning of the SPE cartridge. As a result, purees can be extracted without previous enzymatic treatment, as it is required by other methods that use multiple liquid-liquid extractions. Patulin was well separated from the main interfering compound 5-hydroxymethylfurfural during chromatography when using RP-HPLC columns that allow the separation of rather polar substances with mobile phases of more 99% of water. Additionally to this method A and due to the large number of laboratories that intended to participate in the validation process, the participants were split into two groups and a second method B was validated. This method B is a slightly modified version with the same principle as the one previously published by MacDonald et al. in 2000. The main modifications related to the aliquotation. Patulin is extracted three times from the juice or the de-pectinated puree with neat ethyl acetate. The combined ethyl acetate phases were re-extracted with sodium carbonate solution and evaporated. The residue was then re-dissolved in 0.1 % acetic acid solution and separated by HPLC as in method A. Both methods were tested for the determination of patulin in apple juice and fruit puree at the proposed European regulatory limit of 10 µg/kg. The methods were validated in an international interlaboratory trial in which laboratories from EU Member States, Japan and Brazil participated. Method performance parameters for both apple juice and fruit puree were calculated based on results for spiked samples blind duplicates at two levels and based on results for naturally contaminated samples blind duplicates at three levels for both methods. Test portions of the samples were spiked at levels of 10 µg/kg and 25 µg/kg patulin for both apple juice and fruit puree. In conclusion, the new developed method A showed acceptable within-laboratory and between-laboratory precision for both juice and puree at all levels, while method B only fulfilled partially the performance parameters as required by current EU legislation. Therefore, the newly developed method allows the enforcement of EU legislative limits for patulin in foods for infants at 10 µg/kg. Finally the development and in-house validation of a method for determination of patulin using ultra high pressure liquid chromatography in combination with a mass selective detector, resulting in a better chromatographic and analyte selective separation within a shorter time is described. This is of interest especially for projects in which larger amounts of results need to be generated. A survey with more than 200 samples of baby foods, apple purees and tomato purees from the EU food market was performed with this method. It indicated that during the period of 2006 almost all products were free of patulin and all products were compliant with current EU legislation.

La calidad de los alimentos consumidos por el ser humano y por los animales está determinada, entre otros, por dos factores que tienen lugar al inicio de la cadena de producción. Por un lado, la expansión agrícola influye como consecuencia del gran número de plaguicidas que se utilizan en la agricultura como herramientas para el control de plagas y para incrementar los rendimientos de los cultivos. Por otro lado, los procesos de intensificación de la producción animal requieren de disponer gran cantidad de alimentos conservados que se almacenan en condiciones muy variables y son propensos al desarrollo fúngico y la producción de un gran número de micotoxinas. La combinación de ambos, los plaguicidas y las micotoxinas, puede afectar no solamente la salud y la producción de los animales que son los primeros expuestos, sino eventualmente también a las personas que consumen alimentos producidos por esos animales, como la leche. El control de materias primas y productos en la etapa inicial de la cadena de producción es, por lo tanto, de importancia fundamental.Las actividades desarrolladas para la concreción de la presente tesis estuvieron enmarcadas en dos proyectos de investigación interdisciplinares. Uno denominado ?Residuos y contaminantes en cadenas de agroalimentos prioritarias de la región central santafesina? (Proyecto ANPCyT-PICT) cuyo objetivo principal era generar y perfeccionar métodos avanzados para la determinación de plaguicidas y contaminantes fúngicos, y aplicarlos en estudios de residualidad en muestras de leche cruda y alimentos vegetales para ganado. El otro fue un proyecto de cooperación binacional Argentina-República Checa denominado ?Técnicas analíticas innovadoras para la evaluación de inocuidad de alimentos y piensos? (Proyecto MINCyT-MEYS) uno de cuyos objetivos era la aplicación de metodologías multi-residuo multi-clase de amplio espectro para efectuar estudios de campo sobre la transferencia de micotoxinas y plaguicidas desde los alimentos consumidos por el ganado hacia la leche.En ese contexto, luego de una introducción general el presente manuscrito se organizó en 4 capítulos principales. Los Capítulos 2 y 3 tienen una componente principal de índole químico-analítica centrada en la matriz leche destinada a la obtención de métodos multi-residuo multi-clase para la determinación de micotoxinas y plaguicidas en ese alimento y su aplicación en un estudio de campo (Proyecto ANPCyT-PICT). Los Capítulos 4 y 5, centrados en piensos como matrices de estudio, no focalizan en el desarrollo de metodologías analíticas sino en la aplicación de métodos de amplio espectro para evaluar la incidencia de la residualidad de dichos contaminantes en una diversidad de alimentos que componen la dieta del ganado lechero en la zona central de Argentina (Proyecto MINCyT-MEYS).El Capítulo 2 describe, en una primera parte, la optimización y validación de una metodología basada en columnas de inmunoafinidad con cromatografía líquida acoplada a espectrometría de masa (IAC-UHPLC-MS/MS) para la determinación de aflatoxina M1 en leche y su aplicación para el análisis de 160 muestras de leche tomadas de 40 establecimientos productores de la zona central de la Provincia de Santa Fe durante 4 estaciones climáticas a lo largo de un año. Se encontró una prevalencia de AFM1 en las muestras analizadas cercana al 50% con niveles medios de concentración de 0,038 y un máximo de 0,293 µg/L. Aunque no se registraron excesos en el límite máximo establecido en Argentina (0,5 µg/L) se verificaron violaciones al LM de la Unión Europea (0,05 µg/kg) en el 8% de las muestras. En la segunda parte del Capítulo se describe la optimización de una metodología alternativa al método IAC basada en el enfoque QuEChERS para integrar la determinación de AFM1 con la de residuos de plaguicidas en un mismo método de extracción. Tras varias etapas de optimización, el método propuesto mostró un desempeño analítico con cifras de mérito compatibles con las normativas de Mercosur y la Unión Europea.El Capítulo 3 describe la optimización y validación de una metodología QuEChERS-UHPLC-MS/MS para la determinación multi-residuo multi-clase de residuos de plaguicidas y micotoxinas en leche, y su posterior aplicación en al análisis de 80 muestras de leche tomadas de los mismos establecimientos referidos anteriormente durante dos períodos climáticos. El método optimizado demostró parámetros de desempeño que satisfacen los requerimientos de las normativas de Argentina y de otros países. En el análisis de muestras de leche se encontró mayor prevalencia de insecticidas organofosforados como clorpirifos y diazinon, y en menor medida otros como atrazina, acetamiprid, carbendazim, pirimifos-metilo, entre otros. Adicionalmente, la posterior incorporación de un instrumento GC-MS/MS en las instalaciones del PRINARC permitió analizar los extractos de las mismas muestras ampliando así el conjunto de compuestos determinados. De esa forma, se encontraron también residuos de plaguicidas organoclorados como heptacloro o isómeros de DDT, prohibidos hace más de dos décadas, y una alta ocurrencia de endosulfán-sulfato, de prohibición más reciente en Argentina.En el Capítulo 4 se describe la determinación de residuos de plaguicidas en una amplia variedad de piensos que componen las dietas del ganado lechero. La estrategia analítica orientada a enfoques de amplio espectro consistió en la determinación de 350 compuestos mediante una metodología analítica QuEChERS-UHPLC-MS/MS (análisis realizado en República Checa) y la posterior determinación de 160 plaguicidas en las mismas muestras replicando el procedimiento de extracción pero con determinación por GC-MS/MS (análisis realizado en Argentina). De los 420 plaguicidas estudiados en total 50 se encontraron en las muestras analizadas, siendo el 62% insecticidas, el 20% herbicidas y el 18% restante fungicidas. Clorpirifos, metolacloro y difenilamina fueron los plaguicidas de mayor ocurrencia entre los insecticidas, herbicidas y fungicidas, respectivamente. Respecto al análisis de muestras, en el 72% de las mismas se encontraron residuos de plaguicidas (n=54), siendo los alimentos balanceados y las semillas de algodón las matrices con mayor cantidad de compuestos detectados. La concentración más alta de un plaguicida hallado fue para el insecticida piretroide deltametrina en un alimento balanceado (532 µg/kg).En el Capítulo 5 se describe la determinación de micotoxinas en el mismo set de muestras de alimentos vegetales analizadas en el Capítulo 4 y a partir del mismo procedimiento de extracción, integrando así la determinación simultánea de gran cantidad de compuestos (multi-residuo) de diferente origen (multi-clase). A partir del mismo extracto QuEChERS utilizado en el análisis de residuos de plaguicidas, se determinó un conjunto de 56 metabolitos fúngicos incluyendo micotoxinas tradicionales, conjugadas o enmascaradas, y micotoxinas emergentes. Se encontró que el 100% de las muestras analizadas contenía al menos 1 micotoxina, y del total de compuestos incluidos en el alcance del método se identificaron 38 (68%). Además de las micotoxinas con incidencia histórica en este tipo de alimentos en la región (aflatoxinas, fumonisinas, zearalenona, deoxinivalenol), se hallaron micotoxinas emergentes como eniantinas, toxinas de Alternaria y beauvericina que no habían sido previamente estudiadas. En algunos casos de verificaron excesos a los límites máximos establecidos por la UE para micotoxinas en piensos, como AFB1 en granos de maíz (LM=5 µg/kg), FB1 en gluten de trigo (LM=6000 µg/kg) y ZEA en silaje de maíz (LM=3000 µg/kg). Los resultados obtenidos en este trabajo constituyen un aporte importante para un mayor conocimiento sobre la residualidad de plaguicidas y micotoxinas en la primer etapa de la cadena de producción, y pueden ayudar a asistir a los productores respecto al mejoramiento del manejo de sus cultivos y productos almacenados destinados a la alimentación animal, para perfeccionar los programas de monitoreo y control necesarios para la evaluación de riesgo, y para asegurar las medidas de protección de la salud tanto animal como humana relacionadas con la inocuidad de los alimentos. ; Among several factors, food and feed quality for human and livestock consumption is determined by two main circumstances occurring at the beginning of the food chain. On one hand, agricultural expansion demands a high number of pesticides to be used for crop protection and to improve crops yields and this might impact on food and feed quality. On the other hand, farm feeding intensification in livestock production demands higher amounts of conserved forages and concentrated feeds which may be exposed to fungal development and mycotoxin contamination as a consequence of inappropriate storage conditions. Combination of both pesticide residues and mycotoxins could affect not only animal health and production, but eventually human health also, due to consumption of contaminated products of animal origin. The control of commodities and raw primary foods at the beginning of the food supply chain is, hence, of priority importance. The activities involved in the realization of this thesis where framed under two interdisciplinary research projects. One of them, named "Residues and contaminants in priority agrifood supply chains from Santa Fe central region" (ANPCyT-PICT Project) was aimed to develop and perfect advanced analytical methods for the determination of pesticides and fungal contaminants, and to apply them in residuality studies on raw milk and feedstuff samples for dairy cattle. The second project named "Novel analytical techniques for food and feed safety assessment" was a binational cooperation project between Argentina and the Czech Republic and was aimed to the application of wide-scoped multi-residue multi-class methodologies for field studies assessments regarding mycotoxin and pesticide transfer from feed consumed by dairy cattle to milk. In this context, this manuscript was organized in 4 main chapters after a general introduction about the different addressed topics. Chapters 2 and 3 have a main chemical-analytical component focused on milk as the studied matrix and are aimed to the development of multi-residue multi-class methods for the determination of mycotoxins and pesticide residues with further application to field studies (ANPCyT-PICT Project). Centered in vegetal origin feedstuffs analysis, Chapters 4 and 5 are not focused on analytical methodologies developments but on their application of wide-scoped approaches to assess mycotoxin and pesticide residuality on a great variety of typical feed that constitute dairy cattle diets in Argentinean central regions (MINCyT-MEYS Project). Chapter 2 describes, at a first stage, the optimization and validation of an immunoaffinity-based extraction method with liquid chromatography-tandem mass spectrometry determination (IAC-UHPLC-MS/MS) for aflatoxin M1 determination in raw milk and its application for the analysis of 160 milk samples obtained from 40 different milk farms located in Santa Fe province central region throughout four climate season in a one-year field study. AFM1 prevalence in the analyzed samples was close to 50% with mean and maximum concentration levels of 0.038 and 0.293µg/L, respectively. Although no maximum limits violations to Argentinean legislation (0.5µg/L) were observed, European Union ML (0.05µg/L) exceedances were observed in 8% of the cases. Chapter 2 second part describes the optimization of a QuEChERS-based approach as an alternative to the IAC method to integrate AFM1 and pesticide residues determination in a single extraction method. After several optimization steps, the proposed method was validated demonstrating appropriate analytical performance to fulfill Mercosur and European Union legislations. Chapter 3 describes the optimization and validation process of a QuEChERS-UHPLC-MS/MS for the multiresidue multi-class determination of pesticides and mycotoxins in milk, and its later application to the analysis of 80 milk samples obtained from the same above mentioned dairy farms. The optimized method demonstrated good performance characteristics to fulfill analytical requirements set by legislation from Argentina and other countries. The analysis of milk samples revealed a higher prevalence of organophosphate insecticides (mainly chlorpyrifos and diazinon) and to a less extent other compounds such as atrazine, acetamiprid, carbendazim and pirimifos-methyl were also found. In addition, the later incorporation of a GC-MS/MS system to the PRINARC analytical platforms allowed the analysis of the same sample extracts to enhance the determined compounds scope. Thus, residues of some organochlorine pesticide that have been banned for over 2 decades were found such as heptachlor and DDT isomers, and also high occurrence of endosulfan-sulfate which has been more recently banned. Chapter 4 describes the pesticide residues determination in a wide variety of feed samples used in dairy cattle diets. In this case the analytical strategy was focused on wide-scoped approaches and it implied the determination of 350 compounds my means of a QuEChERS-UHPLC-MS/MS analytical methodology (analysis performed in the Czech Republic) together with the further determination of 160 pesticides in the same samples by replicating the extraction procedure but with final determination by GC-MS/MS (analysis performed in Argentina). From the total 420 different pesticides studied 50 of them were found in the analyzed samples, being 62% of them insecticides, 20% herbicides and 18% fungicides. Chlorpyrifos, metholachlor and diphenylamine were the pesticides with major occurrence from the insecticide, herbicide and fungicide families, respectively. Regarding sample analysis, pesticide residues were found in 72% of them (n=54), being compound feeds and cotton seeds the feed types with higher amount of detected compounds. The highest concentration of a single pesticide in an individual sample was due to the pyrethroid insecticide deltamethrin in a compound feed sample (532µg/kg). Chapter 5 describes the mycotoxin determination in the same set of feed samples analyzed in Chapter 4, using the same extraction procedure thus integrating simultaneous determination of a great amount of compounds (multi-residue) from different classes and origin (multi-class). From the same sample extracts used for pesticide residues analysis, 56 fungal origin metabolites were determined, including traditional mycotoxins, conjugated or masked mycotoxins and emerging mycotoxins. All of the analyzed samples (100%) evidenced contamination with at least one mycotoxin, and from the total compound included in the analytical scope 38 different mycotoxins were identified (68%). Besides mycotoxins with historical incidence in this types of feed from the studied region (like aflatoxins, fumonisins, zearalenone and deoxynivalenol), other emerging mycotoxins that had not previously been studied were found, such as enniatins, Alternaria toxins and beauvericin. In some cases violations to EU maximum levels for mycotoxins in feeds were observed, as the case of AFB1 in maize grains (ML=5 µg/kg), FB1 in wheat gluten (ML=6000 µg/kg) and ZEA in maize silage (ML=3000 µg/kg). The results obtained from this work constitute an important contribution for a better knowledge about mycotoxin and pesticide residuality in the first stage of the food production chain, and can help to assist producers on the improvement of crops and feed managements, to improve monitoring and control programs for risk assessment and to guarantee safe food and feeds for human and animal health protection. ; Fil: Michlig, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Programa de Investigación y Análisis de Residuos y Contaminantes Químicos; Argentina

Testing for embryotoxicity in vitro is an attractive alternative to animal experimentation. The embryonic stem cell test (EST) is such a method, and it has been formally validated by the European Centre for the Validation of Alternative Methods. A number of recent studies have underscored the potential of this method. However, the EST performed well below the 78% accuracy expected from the validation study using a new set of chemicals and pharmaceutical compounds, and also of toxicity criteria, tested to enlarge the database of the validated EST as part of the Work Package III of the ReProTect Project funded within the 6th Framework Programme of the European Union. To assess the performance and applicability domain of the EST we present a detailed review of the substances and their effects in the EST being nitrofen, ochratoxin A, D-penicillamine, methylazoxymethanol, lovastatin, papaverine, warfarin, +¦-aminopropionitrile, dinoseb, furosemide, doxylamine, pravastatin, and metoclopramide. By delineation of the molecular mechanisms of the substances we identify six categories of reasons for misclassifications. Some of these limitations might also affect other in vitro methods assessing embryotoxicity. Substances that fall into these categories need to be included in future validation sets and in validation guidelines for embryotoxicity testing. Most importantly, we suggest conceivable improvements and additions to the EST which will resolve most of the limitations.