Scientific fields are always changing, and it is important to determine the directions in which they are heading as plans are made for future research activities and research results transition to applications and policies. The MycoKey project, an EU-funded Horizon 2020 project, includes a series of "Roundtable Discussions" to gather information from leading researchers inside and outside the project on directions in which the field of mycotoxicology is trending. This presentation includes a summary of the roundtable discussion on priorities and critical issues in chemical detection and monitoring of mycotoxins. The goal was to identify key elements that could impact when and how methods are used to identify mycotoxins and to increase food safety. The discussion was managed by using the Nominal Group discussion Technique (NGT). The NGT provides for equal input from all participants and is well-known as a process for generating a large number of ideas, while also providing a mechanism for ranking them. The discussion was focused on four following key questions: oto identify scientific and technological innovations which hold promise for development/improvement in the future (question 1);oto identify and prioritize critical elements making methods effectively applicable in -industry for autocontrol and HACCP (question 2), -developing countries with limited analytical capabilities (question 3), -official control with particular reference to multi-mycotoxin methods (question 4). The rankings and the total list of responses provided a rich and detailed context from which particular ideas and general trends could be extracted. Test kits, usually antibody based, were one major focus of the discussions because of their many favorable features, e.g., cost, speed, and ease of use. Most important was considered the degree to which antibody-based diagnostics have become an acceptable standard in many practical applications. Much of the discussion was on how to improve these kits and to speed the detection of mycotoxins in locations where access to a well-equipped analytical chemistry laboratory is neither timely nor cost-effective. The second area of focus for this discussion was multi-mycotoxin detection protocols and challenges remaining before these protocols become methods of choice for regulated mycotoxins. This second point mirrors the first, through the increasing importance of technically highly sophisticated multi-mycotoxin detection protocols. These protocols need more standardization and cross-laboratory validation, but are the future for many official regulatory controls, especially as the number of toxins that are regulated increases. Multi-mycotoxin assays are being developed at a large number of locations and international inter-laboratory validation and comparability of results are needed to establish credibility of results across both scientific and regulatory boundaries. Common critical factors for analyses in academic, industrial, a

Mycotoxins are toxic fungal metabolites that may contaminate several agricultural products, both in the field and during storage. Foodstuffs and feedstuffs contaminated by these natural contaminants may cause serious risks to human and animal health due to their toxic effects.For reliable surveillance programs is mandatory the availability of rapid and cost-effective methods for mycotoxin monitoring that should be validated according to harmonized guidelines. Performance evaluation according to internationally recognized validation programs ensures compliance with regulations and facilitates trade, giving the stakeholders more confidence to choose the proper assay with respect to the application. Development, validation and one-site testing of immunoassays for rapid mycotoxin detection is one of the priority tasks of the MycoKey project (http://www.mycokey.eu/), an EU multidisciplinary project coordinated by the Institute of Sciences of Food Production, National Research Council of Italy (ISPA-CNR), aimed to develop smart solutions to reduce the major occurring mycotoxins in economically important food and feed chains.A brief overview of recent activities carried out at ISPA-CNR on the development and validation of immunoassays for the rapid determination of mycotoxins in cereals and cereal-based products will be presented, with a focus on multi-mycotoxin assays for on-site application. In particular, performances, advantages and limitations of newly developed fluorescence polarization immunoassays and immunochromatographic assays (i.e. lateral flow devices or dipsticks) for the determination of the major mycotoxins occurring in cereals and cereal-based products, as well as the use of DNA aptamers as alternative novel biorecognition agents in biosensor applications for mycotoxins detection, will be presented.

T-2 toxin is a trichothecene mycotoxin produced when Fusarium fungi infect grains, especially oats and wheat. Ingestion of T-2 toxin contaminated grain can cause diarrhea, hemorrhaging, and feed refusal in livestock. Cereal crops infected with mycotoxin-producing fungi form toxin glycosides, sometimes called masked mycotoxins, which are a potential food safety concern because they are not detectable by standard approaches and may be converted back to the parent toxin during digestion or food processing. The work reported here addresses four aspects of T-2 toxin-glucosides: phytotoxicity, stability after ingestion, antibody detection, and the anomericity of the naturally occurring T-2 toxin-glucoside found in cereal plants. T-2 toxin-?-glucoside was chemically synthesized and compared to T-2 toxin-?-glucoside prepared with Blastobotrys muscicola cultures and the T-2 toxin-glucoside found in naturally contaminated oats and wheat. The anomeric forms were separated chromatographically and differ in both NMR and mass spectrometry. Both anomers were significantly degraded to T-2 toxin and HT-2 toxin under conditions that mimic human digestion, but with different kinetics and metabolic end products. The naturally occurring T-2 toxin-glucoside from plants was found to be identical to T-2 toxin-?-glucoside prepared with B. muscicola. An antibody test for the detection of T-2 toxin was not effective for the detection of T-2 toxin-?-glucoside. This anomer was produced in sufficient quantity to assess its animal toxicity.

During the last ten years, Norwegian cereal grain industry has experienced large challenges due to Fusarium spp.and Fusarium mycotoxin contamination of small-grained cereals. To prevent severely contaminated grain lotsfrom entering the grain supply chain, it is important to establish surveys for the most prevalent Fusarium spp. andmycotoxins. The objective of our study was to quantify and calculate the associations between Fusarium spp. andmycotoxins prevalent in oats and spring wheat. In a 6-year period from 2004-2009, 178 grain samples of springwheat and 289 samples of oats were collected from farmers' fields in South East Norway. The grains were analysedfor 18 different Fusarium-mycotoxins by liquid chromatography - mass spectrometry. Generally, the medianmycotoxin levels were higher than reported in Norwegian studies covering previous years. The DNA content ofFusarium graminearum, Fusarium culmorum, Fusarium langsethiae, Fusarium poae and Fusarium avenaceumwere determined by quantitative PCR. We identified F. graminearum as the main deoxynivalenol (DON) producerin oats and spring wheat, and F. langsethiae as the main HT-2 and T-2-toxins producer in oats. No association wasobserved between quantity of F. graminearum DNA and quantity of F. langsethiae DNA nor for their respectivemycotoxins, in oats. F. avenaceum was one of the most prevalent Fusarium species in both oats and spring wheat.The following ranking of Fusarium species was made based on the DNA concentrations of the Fusarium spp.analysed in this survey (from high to low): F. graminearum = F. langsethiae = F. avenaceum > F. poae > F. culmorum(oats); F. graminearum = F. avenaceum > F. culmorum > F. poae = F. langsethiae (spring wheat). Our results are inagreement with recently published data indicating a shift in the relative prevalence of Fusarium species towardsmore F. graminearum versus F. culmorum in Norwegian oats and spring wheat.

In this study, the naturally debittered table olives cv Bella di Cerignola were studied in order to (i) characterizetheir phenolic composition; (ii) evaluate the polyphenols bioaccessibility; (iii) assess their absorption and transport, acrossCaco2/TC7. LC-MS/MS analysis has confirmed the presence of hydroxytyrosol acetate, caffeoyl-6?-secologanoside, andcomselogoside. In vitro bioaccessibility ranged from 7% of luteolin to 100% of tyrosol, highlighting the flavonoids sensitivity tothe digestive conditions. The Caco2/TC7 polyphenols accumulation was rapid (60 min) with an efficiency of 0.89%; the overallbioavailability was 1.86% (120 min), with hydroxytyrosol and tyrosol the highest bioavailables, followed by verbascoside andluteolin. In the cells and basolateral side, caffeic and coumaric acids metabolites, probably derived from esterase activities, weredetected. In conclusion, the naturally debittered table olives cv Bella di Cerignola can be considered as a source of bioaccessible,absorbable, and bioavailable polyphenols that, for their potential health promoting effect, permit inclusion of table olives as afunctional food suitable for a balanced diet.

The main objective of the present study was the characterization of phenolic compounds from "Bella di Cerignola" table olive cultivar, one of the most important variety in Apulia (Southern Italy), that received the "Protected Denomination of Origin" (DOP) in 2000 by EU. In addition, the assessment of bioaccessibility of these polyphenols was investigated by using in vitro gastro- intestinal digestion model1. Methodologies: After extraction, the phenolic concentration was determined by HPLC according to Lattanzio 1982(2). Moreover, the olives were subjected to successive gastric and pancreatic digestion, following the method of Versantvoort et al. (2005)1 .The polyphenols were identified by HPLC-DAD and LC-HRMS analysis in order to assess their bioaccessibility. Results and conclusion: The main polyphenols identified were: hydroxytyrosol, tyrosol, caffeic acid, verbascoside, isoverbascoside, caffeoyl-6-secologanoside, comselogoside, luteolin. Interesting is the presence of comselogoside and caffeoyl-6-secologanoside, already identified in OMWW3 and olive fruits4 . After in vitro digestion process, all total phenols identified were bioaccessible, apart luteolin that was absent in the digestive fraction. In particular, the bio-accessibilities were: 84.2% for hydroxytyrosol, 100% for tyrosol and caffeic acid, 47 % for verbascoside, 75% for isoverbascoside, 77% for caffeoyl-6-secologanoside, 81% for comselogoside. Regarding tyrosol, caffeic acid and isoverbascoside, their amount, in the chyme fraction, was higher than no digested olives, probably for hydrolysis and isomerizations phenomena occurred in the gastro-intestinal conditions. In conclusion, the data obtained provide preliminary insight on the potential for bioavailability of the polyphenols present in a complex matrix such as "Bella di Cerignola" cultivar.

Fusarium verticillioides (teleomorph Gibberella moniliformis) is the main fungal agent of ear and kernel rot of maize (Zea mays L.) worldwide, including Italy. F.verticillioides is a highly toxigenic species since it is able to produce the carcinogenic mycotoxins fumonisins. In this study, 25 F. verticillioides strains, isolated from maize in different regions of Italy were analyzed for their ability to produce fumonisins, their pathogenicity and their genetic variability. A further referenced strain of G. moniliformis isolated from maize in USA was also used as outgroup. The fumonisins B?, B?, and B? were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pathogenicity tests were carried out by symptom observation and determination of growth parameters after inoculation of maize seeds, seedlings and wounded detached leaves. Total genomic DNA was used for Amplified Fragment Length Polymorphism (AFLP) analysis. About 20% of the analyzed strains were unable to produce fumonisins in in vitro experiments on inoculated maize flour, while, among fumonisin producers, a great variability was observed, with values ranging from 1 to 115 mg kg?¹. The different analyzed strains showed a wide range of pathogenicity in terms of effect on seed germination, seedling development and of symptoms produced on detached leaves, which were not correlated with the different in vitro fumonisin production. AFLP analysis indicated the presence of genetic diversity not only between the Italian strains and the American reference but also among the Italian isolates.

The results of a proficiency test for the LC-MS/(MS) determination of up to 11 mycotoxins (aflatoxins B-1, B-2, G(1) and G(2), fumonisins B-1 and B-2, ochratoxin A, deoxynivalenol, T-2 and HT-2 toxins and zearalenone) in maize were evaluated to identify possible strengths and weaknesses of various methodologies used by the 41 participating laboratories. The majority of laboratories (56%) used mixtures of acetonitrile:water for extraction. Other laboratories used methanol:water mixtures (17%) or performed two consecutive extractions with phosphate buffer solution (PBS) followed by methanol (15%). Few laboratories used mixtures of acetonitrile:water:methanol (7%), water:ethyl acetate (2.5%) or PBS alone (2.5%). The majority of laboratories (58%) used a clean-up step prior to chromatography. The remaining laboratories analysed crude extracts (37%) or used a mixed approach (5%). The amount of sample equivalent injected into LC-MS/(MS) ranged between 0.1-303 mg for purified extracts and 0.08-20 mg for directly analysed crude extracts. External (54%), matrix-matched (22%) or stable isotope-labelled internal standards calibration (24%) were used for toxin quantification. In general, extraction mixtures of water with acetonitrile, methanol or both provided good results for quantitative extraction of mycotoxins from maize. Laboratories using sample extract clean-up reported acceptable results for the majority of mycotoxins. Good results were also obtained by laboratories that analysed crude extracts although a high variability of results was observed for all tested mycotoxins. Matrix-matched calibration or isotope-labelled internal standards efficiently compensated matrix effects whereas external calibration gave reliable results by injecting <= 10 mg of matrix equivalent amounts. Unacceptable high recovery and high variability of fumonisin results were obtained by the majority of laboratories, which could not be explained and thus require further investigation. These findings provide the basis for the optimization and selection of methods to be used in future interlaboratory validation studies to derive their performance characteristics for simultaneous determination of mycotoxins in maize.

Safety of milk may be affected by toxic contamination. Aflatoxin B1 (AFB1) is a secondary metabolite produced by some Aspergillus spp. fungi affecting many crops and feed materials. Milk is the only food of animal origin where a significant aflatoxin feed-food carry over may occur. The main AFB1-related compound present in milk is aflatoxin M1 (AFM1), the 4-hydroxylated metabolite of AFB1, classified by the IARC as "group 1 human carcinogen". Regulation No. 1881/2006/EC established the maximum level for AFM1 in raw milk, heat-treated milk and milk for the manufacture of dairy products at 50 ng/kg. Scientific and technological research is called to develop cost- and time-effective field methods that can be transferred to producers for self-control purposes. When validated according to official guidelines,such as those defined in the Commission Regulation 519/2014/EU, rapid methods are expected to complement the consolidated European system for official control that is actually based on sophisticated and expensive laboratory instruments and techniques requiring extensive sample pre-treatment and personnel training.Aim of this work was to evaluate and compare analytical performances of widely applied commercial immunoassays for the detection of AFM1 in raw cow milk, namely lateral flow immunoassay (LFD) and ELISA. Analytical performances such as precision profile, cuf-off, false positive and false negative rates were evaluated for each assay by single laboratory validation performed at AFM1 levels of 0, 25, 50 and 75 ng/kg. Fifty ng/kg was the screening target concentration (STC), that was the concentration of interest for the detection of the mycotoxin in the milk sample. The two assays showed similar performances in terms of cut-off (37.7 ng/kg and 39.4 ng/kg for LFD and ELISA respectively), false suspects rate for blanks (< 0.1% for both assays) and false negative rate for samples containing AFM1 at levels higher than STC (0.3% for both assays). False suspect rate for samples contaminated at 25 ng/kg (50% STC) were 3% and 23% for LFD and ELISA respectively. Similar values were also obtained for the precision at all tested validation levels which was < 19% for the LFD and < 24 %for the ELISA.Furthermore, a satisfactory correlation of the results obtained with the rapid immunoassays and the AOAC Official Method 2000.08 was obtained for the analysis of cow milk samples naturally contaminated at AFM1 levels in the range n.d. - 50 ng/kg. Finally, the extension of the scope of the LFD method to goat and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation. The obtained data showed the applicability of the LFD immunoassay to goat and sheep milk provided that a specific calibration curve was used.

Results obtained within a project undertheEuropean Commission standardization mandateM520/EN (item 4) are herein presented. Aim of theproject was to develop and validate an analyticalmethod for the simultaneous determination ofnivalenol, deoxynivalenol and its acetyl derivatives,T-2 and HT-2 toxins, and zearalenone in cereals andcereal products by liquid chromatography - tandemmass spectrometry. The standardized method shallbe applied to perform official control of the abovefood products, and to determine whether aproduction batch can be put on the market.

For enforcement purposes and forreliable surveillance programs, the availability ofvalidated methods for determining foodcontaminants with performance characteristics thatmeet certain minimum criteria is mandatory. A briefoverview of recent activities carried out at theInstitute of Sciences of Food Production, NationalResearch Council of Italy (ISPA-CNR) on thedevelopment and validation of analytical methodsfor the determination of mycotoxins in food ispresented.

Development, validation and one-site testing of immunoassays for rapid mycotoxin detection is one of the priority tasks of the MycoKey project (http://www.mycokey.eu/), an EU multidisciplinary project, aimed to develop smart solutions to reduce the major occurring mycotoxins in economically important food and feed chains. In this framework, a multiplex dipstick immunoassay based method for the simultaneous quantitative determination of major Fusarium toxins, namely deoxynivalenol (DON), zearalenone (ZEA), and fumonisins (FUM) in cereals (wheat, barley and maize) is under development. The dipstick format is based on an indirect competitive approach. The DON/ZEA/FUM prototype is based on a nitrocellulose lateral flow device using a reader to enable quantitative determination of mycotoxin contamination in cereal extracts. Three test lines (mycotoxin-BSA conjugates) and one control line are located on the strip membrane, whereas labeled antibodies were freeze-dried within a microwell. The prototype strip test development included the following steps: definition of coating process and detection reagent formulation parameters; definition of assay parameters like incubation time, migration time, volume sampling to reach minimum performance requested in term of LoD (limit of detection), LoQ (limit of quantification); development of a sample preparation protocol allowing satisfactory mycotoxin recoveries. The total time of analysis is 25 minutes including pre analytical treatment.Preliminary results showed the DON/ZEA/FUM prototype to be compatible with the minimum acceptable performances, and to be suitable for its application to the analysis of real samples containing the target mycotoxins at levels close to EU regulatory levels.

Metabolomics is a powerful method for comprehensive investigation of metabolite variations in biological systems. Currently liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) represent the most powerful metabolomic platform. Provided that a proper sample preparation is performed, this technology may allow for the detection of thousands of metabolites and therefore may provide a comprehensive view of the metabolome.Untargeted metabolite mass profiles can be used for biological interpretations, however approaches that do not require the identification of the metabolic features should be used with extreme caution because they may lead to false interpretations. The identification of metabolites with a high level of confidence is required in order to improve the meaning of metabolomics in biological systems, such as plant-pathogen interaction, and possible applications.Well-established computational tools and workflows are important for highly reproducible and repeatable metabolomic studies. In addition, knowledge-based workflows for metabolite annotations should be built, integrating information relevant to MS peaks relationships (adducts and neutral losses), MS/MS data, retention time modeling, with biochemical knowledge. Sharing workflows (research data and software) helps to validate the findings reported in publications and, more importantly, lets researchers freely reuse the data as they are or as a reliable basis to move forward. For these reasons, the aim of this study was to develop an integrated and open source platform for LC-HRMS metabolomic studies. The platform enables processing of data from targeted and untargeted LC-HRMS analysis (profiling and compound annotation). The applicability of the developed approach is here demonstrated through a preliminary investigation of the metabolic response of maize induced by Fusarium verticillioides infection in maize kernels.

This review summarises developments in the determination of mycotoxins over a period between mid-2016 and mid-2017. Analytical methods to determine aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes and zearalenone are covered in individual sections. Advances in proper sampling strategies are discussed in a dedicated section, as are methods used to analyse botanicals and spices and newly developed LC-MS based multi-mycotoxin methods. This critical review aims to briefly discuss the most important recent developments and trends in mycotoxin determination as well as to address limitations of the presented methodologies.

The effect of processing on mycotoxin content in milling fractions has been investigated in 10 samples of durum wheat contaminated with T-2 and HT-2 toxins at levels ranging from 97 to 5,954 ?g/kg (sum of T-2 and HT-2 toxins). Either naturally contaminated samples or samples artificially inoculated with Fusarium sporotrichioides under field conditions were used. A method based on liquid chromatography-tandem mass spectrometry coupled with immunoaffinity column cleanup was validated in-house for the simultaneous analysis of both toxins in a variety of matrices, including uncleaned wheat, cleaned wheat, screenings, bran, red dog, fine middlings, and semolina. Mean recoveries from samples spiked with T-2 and HT-2 toxins at levels of 100 ?g/kg ranged from 85 to 107%, with relative standard deviations (RSDs) lower than 14%. The milling process led to an increase of T-2 and HT-2 toxin contents up to 13-and 5-fold in screenings and bran, respectively, compared with occurrence in the uncleaned wheat; however, an overall reduction of T-2 and HT-2 toxins by 54% (RSD, 20%) and 89% (RSD, 3%) was observed in cleaned wheat and in semolina, respectively. Copyright © International Association for Food Protection.

The effect of nixtamalization on the content of fumonisins (FBs), hydrolysed (HFBs) and partially hydrolysed (PHFBs) fumonisins in maize was investigated at laboratory-scale. Maize naturally contaminated with FBs and PHFBs was cooked with lime. Starting raw maize, steeping and washing waters and final masa fractions were analysed for toxin content. Control-cooking experiments without lime were also carried out. The nixtamalization reduced the amount of FBs and PHFBs in masa and converted them to HFBs. However, the three forms of fumonisins collected in all fractions amounted to 183%, indicating that nixtamalization made available forms of matrix-associated fumonisins that were then converted to their hydrolysed forms. Control-cooking enhanced FBs and PHFBs reduction, due to the solubility of fumonisins in water during the steeping process, but did not form HFBs. These findings indicate that benefits associated with enhancing the nutritional value of nixtamalized maize are also associated with a safer product in terms of fumonisin contamination.

Grains and grain-based products provide the largest contribution to the sum of HT-2 and T-2 toxin exposure for humans and their monitoring is of high priority. The European Commission has recently published a recommendation on the presence of T-2 and HT-2 toxins in cereals and cereal products. An indicative level of 50 ?g/kg has been fixed for the sum of T-2 and HT-2 toxins in durum wheat (Triticum turgidum ssp. durum Desf.) and other cereals for direct human consumption. Wheat debranning, a process by which outer kernel layers are removed through both friction and abrasive scouring actions, positively affects several quality features of semolina such as the improvement of milling yield and the reduction of heat damage. Moreover, the same process provides a powerful tool to improve hygienic-sanitary quality of derived products. Aim of the present study was to evaluate the effectiveness of debranning process to reduce the levels of T-2 and HT-2 toxins in treated kernels and milling fractions. A durum wheat sample contaminated with T-2 and HT-2 toxins at level of 122 ?g/kg (sum of T-2 and HT-2 toxins) was subjected to two consecutive debranning steps at laboratory scale, each one of the duration of 30s. Debranned and not-debranned kernels were processed by a pilot milling plant. All samples, including debranned kernels, debranning wastes and milling fractions (semolina, coarse bran, fine bran and fine middlings) were analysed for the sum of T-2 and HT-2 toxins by enzyme-linked immunoassay (Veratox® for T-2/HT-2, Neogen) and by LC-MS/MS after immunoaffinity column clean up. Results showed that the debranning process significantly reduced levels of T-2 and HT-2 toxins in the kernels with a reduction rate of 72% after the first 30s step. No significant toxin reduction was observed after the second 30s debranning step. High mycotoxin levels were found in the debranning wastes. The semolina fractions, after 0.4 mm sieving, obtained from both debranned and unprocessed kernels showed not significant differences in toxin content, suggesting an accumulation of these toxins in the most peripheral layers. Interestingly, coarse bran obtained from debranned kernels at first step (30s) resulted to be less contaminated (201 µg/kg) than that obtained from unprocessed kernels (433 µg/kg). Preliminary results obtained in the present study indicate that the debranning process is useful to reduce T-2 and HT-2 toxin contamination present on the surface of the wheat kernel and could play an important role to obtain whole-wheat products or less refined products with high nutritional value and hygienic-sanitary quality.

I cereali e i prodotti a base di cereali costituiscono la maggiore fonte di esposizione dell'uomo alle tossine T-2 e HT-2, metaboliti tossici prodotti da funghi del genere Fusarium. Lo scopo del presente lavoro è stato quello di valutare l'effetto del processo di decorticazione sul contenuto delle tossine T-2 e HT-2 nella granella e nelle frazioni di macinazione del frumento duro. Le analisichimiche hanno evidenziato un'efficace azione del processo di decorticazione nel ridurre i livelli di tossine T-2 e HT-2 nella granella e nelle principali frazioni di scarto di macinazione ad elevato valore nutritivo (crusca e farinetta), suggerendo una distribuzione delle due micotossine limitata ai tegumenti esterni delle cariossidi. I livelli di contaminazione nelle semole setacciate ottenute sia dai campioni decorticati, sia tal quali, sono risultati trascurabili.

Rapid methods are nowadays recognized as a strategic tool for mycotoxin issues management. Specific guidelines for validation and verification of mycotoxin screening methods are set in Commission Regulation (EU) No 2014/519. This regulation establishes that the "aim of the validation is to demonstrate the fitness-for-purpose of the screening method" and focuses the entire validation procedure on the determination of specific cut-off levels ensuring a maximum rate of false-negative results of 5%, and the assessment of the rate of false-suspect (positive) results. With regards to rapid test-kits, 'fitness for purpose' includes not only the criteria more commonly considered when discussing laboratory-based methods (specificity, accuracy and precision), but also more "practical" parameters such as speed and ease to implementation in a new operational environment. The latter means demonstrating under local conditions that performance parameters as established during the validation, can be achieved by first time (unskilled) users. This goal can be achieved through "method verification".The aim of the present study was the verification of fitness for purpose of mycotoxin screening methods when applied by first time users. This was done in one laboratory facility, with multiple technicians attending, through results of a training course. The verification study was organized similar to a collaborative exercise, involving 10 groups comprised of two technicians that used the methods for the first time. Four different screening methods were applied, namely fast-ELISA, lateral flow device (LFD), fluorescence polarization immunoassay (FPIA), liquid chromatography - high resolution mass spectrometry (LC-HRMS), for deoxynivalenol screening in wheat and two screening methods (LFD and LC-HRMS) for aflatoxin determination in maize. The results of analyses were used to calculate precision, cut-off values and rate of false suspect results. The obtained performance characteristics were interpreted in terms of correct classification of samples as negative and suspect-positive and by comparing the results of the statistical evaluation with the results from previously performed validation studies. Finally, the statistical analysis of the results (ANOVA) allowed to draw some consideration on major factors affecting method precision.

The aim of this study was the evaluation of compatibility of mixtures of pesticides with white wine matrix in order to establish that all the criteria for a candidate reference material were met (homogeneity, stability, definition of certified property values and related uncertainties).The matrix selected for this assay was a pesticide free white wine (i.e. containing selected pesticides at levels lower than the relevant limit of detection, L.O.D.).The spiking mixtures to be included in the matrix were composed by a selection of pesticides usually found in viticulture, belonging to different chemical classes and characterized by specific mode of action.According to CIPAC Method MT 46, the matrices spiked with pesticide mixtures were stressed at 54°C for 14 days in order to investigate on the achievable long-term stability.Pesticide mixtures were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) evaluating the intrinsic amenability to this analytical technique for every single compound and taking into account data of annex B of European EN 15662:2009 "Foods of plant origin - Determination of pesticide residues using GC-MS and/or LC-MS/MS following acetonitrile extraction/partitioning and clean-up by dispersive SPE - QuEChERS-method".White wine is a high water content matrix (80-100%) and contains a variable amount of carbohydrates and organic acids. Therefore extraction and clean-up were performed according to QuEChERS procedure, according to annex C of EN 15662:2009.The quantitative determination of pesticides in the wine matrix mixture was accomplished by LC-MS/MS and LC-HRMS based in Orbitrap technology. Tuning of instrumental parameters for the multiresidue method, either LC-MS/MS and LC-HRMS settings, was performed by direct infusion of every single pesticide.Data were acquired in Selected Reaction Monitoring mode for LC-MS/MS and HR Full Scan mode for LC-HRMS.In order to fulfill the requirements specified in the document SANCO 10684/2013 (Method validation and quality control procedures for pesticide residues analysis in food and feed), two product ions were selected for the LC-MS/MS method, whereas mass accuracy < 5 ppm was the target for LC-HRMS detection.

A Lactobacillus plantarum fermentation product (Bio21B), obtained after strain growth (14 h) in a wheat flour-based medium, was applied in the bread-making process as taste enhancer, in order to obtain a yeast-leavened bread with reduced salt content (20% and 50%) with respect to a reference bread (REF) not containing the fermentation product. Sensory analysis indicated that the Bio21B bread with salt reduced by 50% had a pleasant taste similar to the salt-containing bread (REF). L32 Glutamate and total free amino acid content did not differ between REF and Bio21B breads, while the acids lactic, acetic, phenyllactic, 4-OH-phenyllactic and indole-3-lactic were present only in Bio21B breads. Moreover, the presence of several umami (uridine monophosphate, inosine monophosphate, adenosine, and guanosine) and kokumi (gamma-L-glutamyl-L-valine) taste-related molecules was ascertained both in REF and in Bio21B breads. Therefore, a possible role of the acidic molecules in compensating the negative perception of salt reduction can be hypothesized.

Trichothecene mycotoxins occur in cereal grains and are produced by different Fusarium species, which may develop in the field, principally in wheat, maize, barley and oats. Trichothecene glucosylation can be included among naturally occurring mechanisms in cereals that lead to a reduction of native mycotoxin accumulation in grains. Although toxicological data are scarce, these modified forms may represent a potential food safety concern because they are not detectable by standard approaches and may be converted back to the parent toxin during digestion or food processing [1]. With respect to type-A trichothecenes, the recently issued Recommendation No. 165/2013/EC of the European Commission, setting indicative maximum permitted levels for T-2 toxin (T-2) and HT-2 toxin (HT-2), also takes into account the increasing knowledge made available on T-2/HT-2 glucosides stating that 'attention should be paid to the occurrence of masked forms, in particular their glycosylated-conjugates'.The increasing instrumental availability and technological advances in LC-MS, often allowing compound identification without the need for reference standards, have had a strong impact on the know how about formation and characterization of modified mycotoxins. Detection of known or "novel" trichothecene-related compounds in fungal cultures, plant material or processed foods can provide insights in detoxification and metabolisms of trichothecenes. Furthermore, the direct determination of free and conjugated forms might enable a more accurate evaluation of the risk of exposure.Recent advances in applications of LC-MS techniques for chemical characterization and quantitative determination of type-A trichothecene glucosyl derivatives will be reviewed and discussed. High-resolution mass spectrometry (HRMS), based on Orbitrap technology, has been applied to investigate on the presence of T-2 and HT-2 glucoside derivatives in naturally contaminated cereals (including wheat, oats and barley) and Fusarium fungal cultures. Molecular structure details obtained by measuring exact masses of main characteristic fragments, with high mass accuracy led to the identification of a monoglucoside derivative of T-2 toxin and two monoglucoside derivatives of HT-2 toxin. The analysis of their fragmentation patterns provided evidence for glucosylation at C-3 position for T-2 toxin and at C-3 or C-4 position for HT-2 toxin [2].Two mono-glucosyl derivatives of neosolaniol (NEO) and one mono-glucoside derivative of diacetoxyscirpenol (DAS) have been identified and characterized by liquid chromatography tandem mass spectrometry (LC-MS/MS). These compounds were detected either in fungal cultures or in cereal samples naturally contaminated with the parent toxins [3]. Furthermore, a preliminary screening for the presence of these modified forms of mycotoxins, carried out on naturally contaminated cereal samples (i.e. wheat, oats and barley) showed a widespread occurrence of type-A trich

The presence of glucoside derivatives of T-2 and HT-2 toxins (type A trichothecene mycotoxins) in naturally contaminated wheat and oats is reported for the first time. The use of advanced high-resolution mass spectrometry based on Orbitrap technology allowed to obtain molecular structure details by measuring exact masses of main characteristic fragments, with mass accuracy lower than 2.8 ppm (absolute value). A monoglucoside derivative of T-2 toxin and two monoglucoside derivatives of HT-2 toxin were identified and characterized. The analysis of their fragmentation patterns provided evidence for glucosylation at C-3 position for T-2 toxin and at C-3 or C-4 position for HT-2 toxin. A screening for the presence of these new masked forms of mycotoxins was carried out on a set of naturally contaminated wheat and oats samples. On the basis of peak area ratio between glucoside derivatives and free T-2 and HT-2 toxins, the presence of glucoside derivatives was more likely in wheat than in oats samples. The present work confirms the widespread occurrence of trichothecene glucosides in cereal grains naturally contaminated with the relevant unconjugated toxins, thus suggesting the importance of developing suitable analytical methods for their detection. Besides toxicity studies, tracking down these new masked forms of trichothecenes along the food/feed chain would enable to collect information on their relevance in human/animal exposure to mycotoxin risk.

The aim of the current study was to improve the antifungal activity of eight lactic acid bacterial (LAB) strains by the addition of phenylpyruvic acid (PPA), a precursor of the antifungal compound phenyllactic acid (PLA), to a defined growth medium (DM). The effect of PPA addition on the LAB's antifungal activity related to the production of organic acids (PLA, D-lactic, L-lactic, acetic, citric, formic and 4-hydroxy-phenyllactic acids) and of other phenylpyruvic-derived molecules, was investigated. In the presence of PPA the inhibitory activity (expressed as growth inhibition percentage) against fungal bread contaminants Aspergillus niger and Penicillium roqueforti significantly increased and was, even if not completely, associated to PLA increase (from a mean value of 0.44 to 0.93 mM). While the inhibitory activity against Endomyces fibuliger was mainly correlated to the low pH and to lactic, acetic and p-OH-PLA acids. When the PCA analysis based on data of growth inhibition percentage and organic acid concentrations was performed, strains grown in DM+PPA separated from those grown in DM and the most active strains Lactobacillus plantarum 21B, Lactobacillus fermentum 18B and Lactobacillus brevis 18F grouped together. The antifungal activity resulted to be strain-related, based on a different mechanism of action for filamentous fungi and the yeast and was not exclusively associated to the increase of PLA. Therefore, a further investigation on the unique unidentified peak in HPLC-UV chromatograms, was performed by LC-MS/MS analysis. Actually, full scan mass spectra (negative ion mode) recorded at the retention time of the unknown compound, showed a main peak of m/z 291.0 which was consistent with the nominal mass of the molecular ion [M-H]- of polyporic acid, a PPA derivative whose antifungal activity has been previously reported (Brewer et al., 1977). In conclusion, the addition of PPA to the growth medium contributed to improve the antifungal activity of LAB strains and resulted in the production of the polyporic acid, here ascertained in LAB strains.

Summary: European Union Regulation 519/2014/EU specifies performance requirements for screening methods for mycotoxins and can be applied also to mass spectrometry approaches. In this work a liquid chromatography high-resolution mass spectrometry method for the simultaneous determination of the major Fusarium toxins in wheat (deoxynivalenol, 3- and 15-acetyl deoxynivalenol, T-2 and HT-2 toxins, zearalenone, enniatins A, A1, B, B1, and beauvericin) was successfully developed and validated according to this regulation.

A strong trend toward using highly selective mass spectrometry technologies for screening of multiple mycotoxins has been observed in recent years. In the present study, the process of validation of a multimycotoxin screening method based on liquid chromatography-high-resolution mass spectrometry method is presented. The method was intended for the simultaneous screening of the major Fusarium toxins (deoxynivalenol, 3- and 15-acetyl deoxynivalenol, T-2 and HT-2 toxins, zearalenone, enniatins A, A1, B, and B1, and beauvericin) in wheat. The sample preparation protocol was based on a double extraction (methanol followed by acetonitrile/water mixture) and purification through solid-phase extraction C18 column. To provide insights for full exploitation of the potential of the double-stage high-resolution mass spectrometry detection, a full-scan acquisition event followed by a sequence of 5 fragmentation events (variable data-independent acquisition) was set for mycotoxin detection, the latter to be exploited for confirmatory purposes. Method analytical performances were evaluated through in-house validation and small-scale interlaboratory study, designed according to Commission Regulation 519/2014/EU, setting performance requirements for screening methods for mycotoxins. Screening target concentrations were close to European Union maximum permitted or indicative levels. The in-house validation provided the precision of the response under repeatability conditions and the intermediate precision (both resulting lower than 30%), the cutoff value, and the rate of false suspect results for negative (free of the mycotoxin of interest) samples, which resulted lower than 0.1% in all cases. The collaborative study provided reproducibility and laboratory independent cutoff values. Analysis of reference materials proved method trueness and suitability for screening of the major Fusarium mycotoxins in wheat. Finally, the applicability of the full-scan/variable data-independent acquisition detection approach was successfully tested on a set of naturally contaminated wheat samples, where 2 characteristic product ions could be detected for all identified mycotoxins even at levels in the low g/kg range.

Mycotoxin contamination of agricultural food commodities and beverages poses a risk to human and animal health due to their toxic effects. Over 100 mycotoxins have been identified, although only a few of them present a significant source of food-borne illnesses and are of major concern worldwide. They are: aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2), ochratoxin A (OTA), fumonisin B1 (FB1) and B2 (FB2), deoxynivalenol (DON), zearalenone (ZEA), T-2 and HT-2 toxins. In the European Union, harmonized maximum levels for mycotoxins in foodstuffs have been specified in the Commission Regulation EC 1881/2006, and further amendments. Effective and efficient analytical methods are therefore required to identify and determine mycotoxins at legislated levels and to enforce regulatory limits. Within this context the application of LC-MS(MS) techniques is being largely explored since it enables the simultaneous monitoring of different mycotoxins in one run. Even though LC-MS(MS) methodologies for single or multiple mycotoxin determination are routinely used in control laboratories, to date none of official or standard methods approved by AOAC International or CEN (European Standardization Committee) is based on LC-MS. Proficiency Testing (PT) is an effective procedure to determine the performance of individual laboratories for specific measurements, providing a clear and a straightforward way of evaluating the accuracy (trueness and precision) of results obtained by different laboratories.An international PT was organized in 2014 to check, next to the laboratory performance, the state-of-art of currently used multi-mycotoxin methods and their implementation in the respective laboratory. The PT was free of charge and was organized by ISPA-CNR in the framework of the Italian project S.I.Mi.S.A. (PON02_00186_3417512) and promoted by the MoniQA Association (www.moniqa.org). Within the EU Network of Excellence MoniQA several efforts have been made for method comparison and deeper understanding of performances of the available LC-MS(MS) methodologies for multiple-mycotoxin analysis [1,2]. Participants were asked to determine DON, FB1, FB2, ZEA, T-2, HT-2, OTA and aflatoxins (AFB1, AFB2, AFG1, AFG2) in maize, and DON, ZEA, T-2, HT-2 and OTA in wheat. The contaminated test materials were produced and characterized by ISPA-CNR. The use of LC-MS(MS) methods, although not strictly required, was highly recommended, while the use of multi-mycotoxin methods was mandatory. Participants were not obliged to determine all toxins in each material, and let free to report only on those mycotoxins that were simultaneously determined with their multi-mycotoxin methodology. Twenty-two participants from 10 countries registered for the exercise. Nineteen laboratories returned 22 sets of results for various combinations of analytes. Three laboratories returned two sets of results obtained by using two different methods for both contaminated maize and wheat. The assign

Mycotoxins are toxic secondary metabolites of filamentous fungi affecting human and animal health. In the European Union harmonized maximum levels for the major mycotoxins occurring in food commodities have been fixed. Reliable analytical methods are therefore required to identify and determine mycotoxins at legislated levels and to enforce regulatory limits. Liquid chromatography-mass spectrometry (LC-MS) methods are being largely used since they enable the simultaneous detection of different mycotoxins. Moreover, they offer several advantages in terms of selectivity, sensitivity, substantial reduction of sample treatment, simultaneous quantification and confirmation of analyte identity. Participation in laboratory proficiency testing (PT) is an essential elements of laboratory quality assurance programmes in relation to mycotoxins analysis. In addition PT can be a useful tool to evaluate the state-of-art of methods for mycotoxin analysis by comparison of laboratory's performances. A number of PT schemes for individual or group of structurally related mycotoxins exist at international level. Only recently the interest is moving toward the organization of PTs for multi-mycotoxin determination by LC-MS. In 2014 the Institute of Sciences of Food Production of the National Research Council of Italy organized an international PT to check, next to the laboratory performance, the state-of-art of currently used multi-mycotoxin methods and their implementation in the respective laboratory. Test materials were maize contaminated with aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2), ochratoxin A (OTA), fumonisin B1 (FB1) and B2 (FB2), deoxynivalenol (DON), zearalenone (ZEA), T-2 and HT-2 toxins, and wheat contaminated with DON, ZEA, T-2, HT-2 and OTA. Eighteen laboratories from 10 Countries using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodologies participated in the study and returned results for various combinations of mycotoxins. The overall performance of participating laboratories and the assessment trend in multi-mycotoxin determination by LC-MS will be presented and discussed.

Fungal growth is the most frequent cause of spoilage in baked goods determining great economic losses. Lactic acid bacteria (LAB) are applied in the process of several bakery products since they significantly contribute to technological and nutritional properties, influence the food flavour and prevent spoilage by producing organic acids, hydrogen peroxide, cyclic dipeptides, bacteriocins, fatty acids, carbon dioxide, ethanol and diacetyl. Among organic acids, lactic, acetic, phenyllactic (PLA) and p-OH-phenyllactic acids (OH-PLA) play a role in inhibiting fungal and bacterial growth [1]. In particular, the inhibitory properties of PLA have been demonstrated against several fungal species. Recently, the research has been moved towards the application of biotechnology aimed to improve PLA production by microorganisms (bacteria and yeast). PLA is a by-product of phenylalanine (Phe) metabolism and its production can be significantly improved by the addition of precursors such as Phe, phenylpyruvic acid (PPA) and ?-ketoglutaric acid to the growth medium. The aim of the current study was to improve the antifungal activity of eight lactic acid bacterial (LAB) strains by the addition of PPA to a defined growth medium (DM) [2]. The effect of PPA addition on the LABs antifungal activity related to the production of organic acids (PLA, D-lactic, L-lactic, acetic, citric, formic and 4-hydroxy-phenyllactic acids) and of other phenylpyruvic-derived molecules, was investigated. In the presence of PPA the inhibitory activity (expressed as growth inhibition percentage) against fungal bread contaminants Aspergillus niger and Penicillium roqueforti significantly increased and was, even if not completely, associated to PLA increase (from a mean value of 0.44 to 0.93 mM).While the inhibitory activity against the yeast Endomyces fibuliger was mainly correlated to the low pH and to lactic, acetic and p-OH-PLA acids. When the Principal Component Analysis (PCA) analysis based on data of growth inhibition percentage and organic acid concentrations was performed, strains grown in DM + PPA separated from those grown in DM and the most active strains Lactobacillus plantarum 21B, Lactobacillus fermentum 18B and Lactobacillus brevis 18F grouped together. The antifungal activity resulted to be strain-related, based on a different mechanism of action for filamentous fungi and the yeast and was not exclusively associated to the increase of PLA. Therefore, a further investigation on the unique unidentified peak in HPLC-UV chromatograms was performed by LC- MS/MS analysis. Full scan mass spectra (negative ion mode) recorded at the retention time of the unknown compound, showed a main peak of m/z 291.0 which was consistent with the nominal mass of the molecular ion [M-H]- of polyporic acid, a PPA derivative whose antifungal activity has been previously reported against 12 species of fungi [3]. Actually, in microorganisms different from LAB, as fungi and lichens, PPA is a precursor

The understanding of mycotoxins transfer to biological fluids is challenged by the difficulties in performing and replicating in vivo experiments as well as the lack of suitable methods of analysis to detect simultaneously a range of chemically different metabolites at trace levels. LC-MS/MS has been used herein to study the urinary excretion profile of the mycotoxin deoxynivalenol in human and Wistar rat. Deoxynivalenol and deoxynivalenol glucuronide were found in both human and rat urines, whereas de-epoxydeoxynivalenol and its glucuronide conjugate were only detected in rat urine. The presence of two deoxynivalenol glucuronide isomers in Wistar rat urine has been shown for the first time. Structure confirmation of the detected metabolites was provided by the analysis of fragmentation patterns. A solid phase extraction clean up procedure allowing recoveries in the range 72-102% for deoxynivalenol, de-epoxydeoxynivalenol, and their glucuronide conjugates was optimized. A multiple reaction monitoring method for the simultaneous determination of all investigated metabolites was elaborated allowing the direct detection of deoxynivalenol metabolites without the hydrolysis step. Deoxynivalenol urinary levels in the range 0.003-0.008. ?g/ml were detected in healthy human subjects, whereas deoxynivalenol and de-epoxynivalenol levels between 1.9-4.9. ?g/ml and 1.6-5.9. ?g/ml, respectively were found in administered rat urine. These findings emphasize the relevance of the highly selective and sensitive LC-MS/MS technique for the direct detection and characterization of deoxynivalenol metabolites in complex biological matrices. © 2011 Elsevier B.V.

A strong trend toward using highly selective high-resolution mass spectrometry technologies for rapid screening of multiple mycotoxins has been observed in recent years. Consequently, within the European Union, specific performance requirements for screening methods for mycotoxins have been issued in the Regulation 519/2014/EU that apply not only to bioanalytical methods based on immuno-recognition, but also to mass spectrometry approaches. This regulation establishes that the "aim of the validation is to demonstrate the fitness for purpose of the screening method", and focuses the entire validation procedure on the determination of cut-off value, false negative and false suspect rates. However, performance characteristics such as sensitivity, selectivity, and precision are embedded in these parameters. In the present study, a liquid chromatography high-resolution mass spectrometry method was developed for the simultaneous determination of the major Fusarium toxins in wheat, namely deoxynivalenol, 3- and 15-acetyl deoxynivalenol, T-2 and HT-2 toxins, zearalenone, enniatins A, A1, B, B1, and beauvericin. A sample preparation protocol was optimized, based on a double extraction (methanol followed by acetonitrile/water mixture), and purification through solid phase extraction C18 columns, achieving satisfactory recoveries for the target mycotoxins covering a wide range of polarities. Analytical performances of the developed method were evaluated through both in house validation and small scale interlaboratory study. The validation study, designed according to the Commission Regulation 519/2014/EU, was performed at screening target concentrations (STC) close to EU maximum permitted or indicative levels. The in house validation procedure provided the precision of the response under repeatability conditions and the intermediate precision (the latter resulting lower than 42% for all mycotoxins), the matrix effect from different wheat varieties on the precision, the cut off value, and the rate of false positive results for samples containing the target mycotoxins at 20% of the STC, that resulted lower than 1% in all cases. A small scale collaborative study was therefore carried out to determine reproducibility and robust and laboratory independent cut off values. Finally, analysis of naturally contaminated samples and reference materials confirmed method suitability for screening of Fusarium mycotoxins in wheat, to check compliance with EU maximum permitted or recommended levels

Cereals and derived products may contain different groups of contaminants, including pesticide residues and mycotoxins, for which maximum limits have been established by the EU legislation. For enforcement purposes it is essential the availability of precise and reliable analytical methods applicable at the regulatory levels. Furthermore a current trend in chemical food safety control is represented by the efforts to integrate analyses of various groups of food contaminants into high-throughput methods with one common sample preparation and a single final determination.Due to increasing availability of analytical equipments enabling high sensitivity and selectivity, contaminants detection and determination by mass spectrometry plays an important role in this field, and sample preparation can become the most challenging task. Several strategies based on either traditional clean up devices or innovative systems are continuously proposed and evaluated for their contribution in the achievement of satisfactory method performances, including acceptable matrix effects. The higher is the selectivity of the sample preparation protocol the lower is the number of analytes that can be included in the method that provides in turn better performances for the specific analytes. On the contrary, less selective clean up strategies, allowing to analyze a larger number of mycotoxins and/or pesticide residues require higher instrumental technical specifications (selectivity and sensitivity) to achieve satisfactory method performances.Despite the large number of currently available LC-MS(/MS) methods for multiple contaminant determination in foods, most of them proved to be suitable for semi-quantitative or screening purposes only due to the lack of sufficient validation efforts. Acceptability criteria for analytical methods are reported in the Commission Regulation 401/2006 for each regulated mycotoxin, whereas criteria for pesticide analysis are reported in the Document SANCO/12495/2013. Another effective procedure for quality assurance and performance verification of the chemical analysis in food control laboratories is represented by Proficiency testing (PT), providing a clear and a straightforward way of evaluating the accuracy (trueness and precision) of results obtained by different laboratories.The application of advanced MS approaches for mycotoxin and/or pesticide control along the cereal food chain will be presented. In particular the following methods will be described, with emphasis to validation aspects according to European standards, and application to real samples:- a method for the simultaneous determination of aflatoxins, ochratoxin A, fumonisins, trichothecenes (including glucosyl and acetyl derivatives) and zearalenone in cereals and derived products based on multi-antibody immunoaffinity column clean up and LC-MS/MS detection;- a method for the simultaneous determination of aflatoxins, ochratoxin A, trichothecenes and zearalenone i

A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin-BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73-109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 ?g kg-1, respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 ?g kg-1, respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC-MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.

Mycotoxin analytical methods in food matrices include rapid screening and confirmation techniques. Development of both quantitative (ELISA, FLISA, FPIA, immunosensors) and qualitative (lateral flow, membrane-based flow-through) systems for (multi)mycotoxin detection is one of the fast-growing trends. Developments of various recognition elements as well as novel sensitive labels are currently ongoing in order to design a reliable and rapid test-system.The Mycokey project aims for the development of different (multi)mycotoxin rapid screening methods. The presentation will highlight the current developments: 1. a multiplex strip test for easy and rapid simultaneous determination of Fusarium toxins (fumonisins, deoxynivalenol and zearalenone) in cereals; 2. DNA aptamer based strip tests for simultaneous analysis of aflatoxin B1 and fumonisinB1 in maize making use of quantum dots as highly sensitive labels; 3. fluorescence polarization immunoassay for the quantitativesimultaneous determination of DON, 3-acetyl-DON, 15-acetyl-DON and DON-3-glucoside in wheat; 4. fluorescence polarization immunoassay for the quantitative simultaneous determination ofT-2, HT-2, T2-glucoside and HT2-glucoside in wheat; and 5. a new biosensor platform for the detection of AFB1 based on real-time electrochemical profiling (REP).Moreover, a MycoKey survey was performed for the collection and sharing of current knowledge and experience on rapid methods for mycotoxin screening. This, together with some lessons learned during the MycoKey round table discussion on mycotoxin detection methods will promote interactive discussions between scientist involved in rapid method development and stakeholders as to provide rapid methods that fit the customers' needs and expectations.

MycoKey, an EU-funded Horizon 2020 project, includes a series of "Roundtable Discussions" to gather information on trending research areas in the field of mycotoxicology. This paper includes summaries of the Roundtable Discussions on Chemical Detection and Monitoring of mycotoxins and on the role of genetics and biodiversity in mycotoxin production. Discussions were managed by using the nominal group discussion technique, which generates numerous ideas and provides a ranking for those identified as the most important. Four questions were posed for each research area, as well as two questions that were common to both discussions. Test kits, usually antibody based, were one major focus of the discussions at the Chemical Detection and Monitoring roundtable because of their many favorable features, e.g., cost, speed and ease of use. The second area of focus for this roundtable was multi-mycotoxin detection protocols and the challenges still to be met to enable these protocols to become methods of choice for regulated mycotoxins. For the genetic and biodiversity group, both the depth and the breadth of trending research areas were notable. For some areas, e.g., microbiome studies, the suggested research questions were primarily of a descriptive nature. In other areas, multiple experimental approaches, e.g., transcriptomics, proteomics, RNAi and gene deletions, are needed to understand the regulation of toxin production and mechanisms underlying successful biological controls. Answers to the research questions will provide starting points for developing acceptable prevention and remediation processes. Forging a partnership between scientists and appropriately-placed communications experts was recognized by both groups as an essential step to communicating risks, while retaining overall confidence in the safety of the food supply and the integrity of the food production chain.

Fusarium langsethiae, formally described as a new species over a decade ago, has been identified as the main producer of HT-2(HT2) and T-2 (T2) toxins in Europe in small cereal grains. Mycotoxin contamination caused by this Fusarium species can representa food safety hazard that deserves further attention. In the present work, the mycotoxin profile in wheat cultures of F.langsethiae is presented with particular reference to the production of major type-A trichothecenes and their glucosyl derivatives.F. langsethiae isolates, representative of the major Italian wheat cultivation areas, were tested for the production of T2,HT2, diacetoxyscirpenol (DAS) and neosolaniol (NEO), and relevant glucosyl derivatives. Liquid chromatography-tandem massspectrometry (LC-MS/MS) was used for the identification and chemical characterization of these metabolites. F. langsethiaeisolates under investigation resulted to be potent producers of T2, HT2 and NEO. Furthermore, a well-defined set of isolates,all originating from Central Italy, produced also DAS. All isolates were found to be able to produce HT2 glucosyl derivatives,whereas only traces of T2 glucoside were detected in one sample. Furthermore, two mono-glucosyl derivatives of NEO and onemono-glucoside derivative of DAS were identified and characterized. The screening for the presence/absence of glucosylatedtrichothecenes in analyzed fungal extracts revealed a general co-occurrence of these derivatives with the parent toxin at levelsthat could be roughly estimated to account up to 37% of the relevant unconjugated toxin. This is the first report of the productionof glucosylated trichothecenes by F. langsethiae cultured on small grains.

Maize is the most important food crop in the world, although it is largely susceptible to mycotoxin contamination causing a negative economic impact and serious health risks to humans and animals. It is well-known that moldy, colored/discolored, injured, broken and damaged grain kernels, as well as dust within a contaminated batch of maize, contain high levels of mycotoxins. A combination of cleaning technologies to efficiently remove contaminated fractions can therefore significantly reduce mycotoxin contamination in the outcome product.Different batches of biomass/feed quality maize contaminated by aflatoxins and Fusarium toxins (namely deoxynivalenol, fumonisins and zearalenone) have been processed on different industrial plants in Italy, Germany and Spain to evaluate the effect of different cleaning solutions on the reduction of mycotoxins. The investigated cleaning solutions included i) mechanical size separation of coarse, small and broken kernels, ii) dust/fine particles removal through an aspiration channel, iii) separation of kernels based on gravity and iv) optical sorting of spatial and spectral kernel defects. A dynamic sampling according to the Commission Regulation No. 401/2006 was performed along the entire process lines. The frequency of sampling was estimated according to the DG-SANCO guidance by taking in account the number of incremental samples, the weight of portion to be sampled (ton) and the unloading speed (ton/hr). A number of incremental samples ranging from 3 to 60 (about 100-300 g each) was collected, depending on the sampled fractions. Mycotoxin analyses of the water-slurry aggregate samples were performed by validated HPLC methods based on immunoaffinity column clean-up of extracts. In addition, in some trials the incoming materials were analyzed by the Eurofin's Rapidust® system for on-site sampling of dusts.A significant reduction of mycotoxin content in the cleaned products was observed for aflatoxins (up to 90%), zearalenone (up to 88%), deoxynivalenol (up to 82%) and fumonisins (up to 69%), with respect to the uncleaned products. High levels of mycotoxins were found in the rejected fractions, with the highest levels in dusts and in the rejected fractions from aspirator and optical sorting.This study shows that a cleaning line combining both mechanical and optical sorting technologies can provide a reliable solution for reducing mycotoxin contamination in maize. In addition, a completely new sorting technology was recently developed by Bühler for grain cleaning and monitoring based on the spectral properties of fluorescence to reduce the risk of aflatoxin contamination in maize.

The occurrence of deoxynivalenol, 3- and 15-deoxynivalenol and deoxynivalenol-3-glucoside in 84 durum wheat samples, from the Argentinean main growing area, was investigated during 2012/13 and 2013/14 using LC-MS/MS. Deoxynivalenol was found in all samples at concentrations varying between <LOQ (50 mu g/kg) and 9480 mu g/kg. Deoxynivalenol-3-glucoside was detected in 94% of the samples at concentrations ranging from <LOQ (50 mu g/kg) to 850 mu g/kg. Moreover, the acetylated derivatives were also detected but at lower frequency (49%). To the best of our knowledge, this is the first report of deoxynivalenol-3-glucoside in wheat in Argentina. All the commercial cultivars transformed deoxynivalenol to its glucosylated form at conversion rates between 6 and 22%. The results obtained alert of the potential risk present in durum wheat for Argentinean consumers but also show that some of the commercial cultivars currently on used could be promising candidates for breeding programs intended to obtained Fusarium head blight resistance. (C) 2017 Elsevier Ltd. All rights reserved.

T-2 and HT-2 toxins are two of the most toxic members of type-A trichothecenes, produced by a number of Fusarium species. The occurrence of these mycotoxins was studied in barley samples during a survey carried out in the 2011-2014 growing seasons in climatically different regions in Italy. The percentage of samples found positive ranges from 22% to 53%, with values included between 26 and 787 µg/kg. The percentage of samples with a T-2 and HT-2 content above the EU indicative levels for barley of 200 µg/kg ranges from 2% to 19.6% in the 2011-2014 period. The fungal species responsible for the production of these toxins in 100% of positive samples has been identified as Fusarium langsethiae, a well-known producer of T-2 and HT-2 toxins. A positive correlation between the amount of F. langsethiae DNA and of the sum of T-2 and HT-2 toxins was found. This is the first report on the occurrence of F. langsethiae--and of its toxic metabolites T-2 and HT-2--in malting barley grown in Italy.

Surveys on the occurrence of type A trichothecenes in wheat, and particularly for the T-2 and HT-2 toxins, and information on the biology and epidemiology of the causative Fusarium species (i.e. F. langsethiae, F. sporotrichioides) are scarce in Italy, as compared to the more common type B trichothecene, deoxynivalenol and its producers. This 4-year monitoring of phytopathogenic Fusarium species on 183 seed lots of durum wheat shows wide distribution of F. langsethiae in Italy and the potential of several isolates of this fungus to produce high amounts of T-2 and HT-2 in wheat. Fusarium langsethiae was observed for approximately 48% of the analysed samples, with a maximum incidence for a single lot of 10.5%. Fusarium sporotrichioides was observed only in 2011, with an average incidence of 2% (range, 0-3%). A collection of F. langsethiae isolates representative of the main cultivation areas in Italy was established. These isolates showed great variability for their toxin production in vitro. Of 28 strains, all except one isolate can produce the T-2 and HT-2 toxins. HT-2 was generally in greater amounts than T-2, with an average concentration ratio for HT-2 to T-2 of 2.1 (range, 0.7-5.4). The artificial inoculation of wheat with three isolates of F. langsethiae produced no Fusarium head blight symptoms under field conditions. However, significantly higher incidence of F. langsethiae was seen on the kernels of inoculated plants, compared to the uninoculated controls.

Seed germination is a key phase of the parasitic plant life cycle that is stimulated by the secondary metabolites, mainly strigolactones (SLs), secreted by the host roots. Interventions during this stage would be particularly suitable for parasitic weed management practices, as blocking these chemical signals would prevent seed germination and thus parasite attack. Four fungal strains with different ecological functions were considered for their possible ability to metabolise SLs: Fusarium oxysporum and F. solani, biocontrol agents of Phelipanche ramosa; Trichoderma harzianum, a potential biopesticide; Botrytis cinerea, a phytopathogenic fungus. Four different SLs [the natural strigol, 5-deoxystrigol (5DS) and 4-deoxyorobanchol (4DO), and the synthetic analogue GR24] were added to fungal cultures, followed by determination of the SL content by liquid chromatography-tandem mass spectrometry.RESULTS: Differences were observed among microorganisms, treatments and SLs used. T. harzianum and F. oxysporum were the most capable of reducing the SL content; considering the whole set of fungi used, 5DS and 4DO proved to be the most degradable SLs.CONCLUSIONS: Beneficial microscopic fungi could differently be used for biocontrolling parasitic weeds, acting as a 'physiological'barrier, by preventing the germination of their seeds through the ability to biotransform the stimulatory signals.

Two Proficiency Testings (PTs) involving eighteen laboratory participants from 10 Countries have been conducted in 2014 for the simultaneous determination of deoxynivalenol, fumonisins B1 and B2, zearalenone, T-2 and HT-2 toxins, ochratoxin A and aflatoxins B1, B2, G1 and G2 in maize and of deoxynivalenol, zearalenone, T-2 and HT-2 toxins and ochratoxin A in wheat, respectively. Theaim of PTs was to check next to the laboratory performance the state-of-art of the LC-MS multimycotoxin methods used by participants. In addition, the trend of performances of LC-MS methods for multi-mycotoxin determination in maize together with method-related issues was assessed by comparing three PTs organized over the years 2011-2014. Data showed the improvement of laboratory performances with the overall acceptable z-scores that progressively increased from 59% in 2011 PT to 85% in 2014 PT, while the rate of unacceptable z-score decreased from 25% in 2011 PT to 11% in 2014 PT.

A proficiency test for simultaneous determination of up to 11 mycotoxins in maize by using LC-MS(MS) methodology was conducted. Purpose of the study was to evaluate the state of the art on the simultaneous determination of mycotoxins by LC-MS(MS) methodology and to obtain information on currently used methodologies and method-related performances. Laboratories were not obliged to determine all target mycotoxins and were free to report only on those mycotoxins that can be simultaneously determined with their LC-MS(MS) methodology.Participants received two test materials: one maize sample naturally contaminated with aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, HT-2 toxin, zearalenone, fumonisins B1 and B2 and one blank maize sample (for spiking). A common mixed mycotoxins calibrant solution and a spiking solution together with spiking protocol, instructions and a comprehensive questionnaire were also distributed. Forty laboratories from fourteen Countries reported results for some combinations of the analytes. In particular, 25 participants analyzed all target mycotoxins whereas the other participants analyzed between 2-10 mycotoxins. Electrospray ionization was used by all laboratories whereas triple quadrupole and ion trap detector was used by 36 and 4 laboratories, respectively. Sample extract cleanup (IMA, SPE, liquid/liquid extraction) was used by 22 participants whereas 17 laboratories analyzed crude extract without purification and 1 participant analyzed the extract with and without purification before LC-MS(MS) determination. For quantification, 10 participants prepared matrix-assisted calibration curves whereas the other 30 participants prepared calibration curves in pure solvents. To compensate matrix effect, 7 of 30 participants using calibration curves prepared in pure solvents added isotope labeled internal standards to the final extract whereas 1 participant used other metabolites (related to zearalenone or deoxynivalenol) as internal standards. Results on the performance of participating laboratories will be presented with particular reference to z-Scores and percent recovery of the 11 mycotoxins.

A liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for the simultaneousdetermination of aflatoxins (B1, B2, G1, G2), ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxinsin wheat flour, barley flour and crisp bread was developed. Mycotoxin fragmentation patterns obtained byhigh-energy collision dissociation (HCD) were investigated to obtain quantitative and confirmatory information(two characteristic masses per mycotoxin) using OrbitrapTM-based high-resolution mass spectrometry.LC-HRMS (full-scan) detection carried out by HCD allows the monitoring of the pseudo-molecular ion andan additional characteristic fragment (for each mycotoxin) with mass accuracy in the range 0.1-3.9 ppm, meetingcurrent European regulatory requirements for LC-MS confirmatory analysis. A sample preparation procedurebased on polymeric solid-phase extraction cartridges was applied, allowing recoveries higher than 74% for ninemycotoxins, with a relative standard deviation lower than 13%. Detection limits in the range 0.5-3.4 mg kg1 wereobtained for three cereal matrices. A critical comparison between the proposed method and a validated methodbased on triple quadrupole mass spectrometry showed similar performance in terms of detection limits, recoveriesand repeatability, and matrix effects. Based on an efficient sample extraction and clean-up, the LC-HCD-HRMSmethod reported here represents a reliable and robust alternative tool for mycotoxin analysis in food matrices ascompared with well-established triple quadrupole-based approaches.

Rapid test methods for measuring Aflatoxin M1 (AFM1) in milk are available either as commercial kits or research methods. Enzyme-lynked immunosorbent assays (ELISA), lateral flow tests, immunoaffinity columns coupled with fluorimetric assay are common formats in the current market. Based on recent research developments it is expected that innovative technologies such as electrochemical affinity biosensors, aptamer based biosensors, dynamic light scattering, might be available in the next future for new kits development.The main purpose of screening methods is to detect the presence of a contaminant at level of interest allowing rapid decision making. European Union has set a maximum permitted limit of 0.050 µg/kg for AFM1 in milk. Besides the high sensitivity required for AFM1 detection in milk, the major analytical challenge when developing screening tests is to make them reliable and robust for laboratory and in-field use. This means to cope with differences from matrix to matrix, environmental conditions, operator skills, lot-to-lot reproducibility. A commonly recognized evaluation system needs to be in place so that commercial kits can be evaluated against the same standard. Evaluation programs for commercial testing kits have been established by the United State Department of Agricultural - Grain Inspection Packers and Stockyards Administration (USDA-GIPSA) and AOAC Research Institute. Recent efforts of the European Union, for establishing practical guidelines for the generation of fit-for-purposes performance parameters for screening methods for mycotoxins in foods, resulted in the Regulation 519/2014/EC. The EC validation scheme and its practical application to evaluate performances of a commercial kit for AFM1 detection in milk will be presented and discussed.

Mycotoxin analytical methods in food matricesinclude rapid screening and confirmation techniques. The amount of publications covering rapid and sensitive on-site tests suitable for non-laboratory simultaneous determination of several analytes in various matrices is constantly rising. Development of both quantitative (ELISA, FLISA, FPIA, immunosensors) and qualitative (lateral flow, membrane-based flow-through) systems for (multi)mycotoxin detection is one of the fast-growing trends. Developments of various recognition elements as well as novel sensitive labels are currently ongoing in order to design a reliable and rapid test-system.Natural receptors such as antibodies are most widely used, but also their synthetic analogues and engineered elements can be employed. Working with complicated matrices or designing of a regenerable test-system can only be realized through the use of stable molecularly imprinted polymers (MIPs) or synthetic peptides. Whereas scFv and scAb fragments or aptamers cannot stand an aggressive environment, they provide high specificity, which is important for analysis of structurally-related compounds. Different labels can be used in immunochemical methods depending on the sample matrix, starting from enzymes (as the most sensitive label) to colloidal gold (widely used). Colloidal semiconductor nanocrystals or quantum dots (QDs) have emerged as a new class of fluorescent labels for biomedical diagnostics, molecular imaging and chemical analysis. The unique optical properties of QDs enable the simultaneous detection of multiple analytes on one single spot provided their conjugates are labeled with QDs which are fluorescent in different parts of the spectrum. Different strategies can be employed for QDs hydrophilization (such as encapsulation with amphiphilic polymer, silica and liposomes) performing different functionalities on the surface and therefore ensuring different conjugation techniques to synthesize QD-labeled immunoreagents.This presentation will give an insight into current trends and innovations in immunochemical methods for (multi)mycotoxin rapid screening. Examples of rapids tests developed in the frame of the Mycokey EU project will be given.

Le micotossine sono sostanze naturali con attività tossica prodotte da diverse specie fungine appartenenti principalmente ai generi Aspergillus, Penicillium e Fusarium. La loro presenza negli alimenti e nei mangimi può essere nociva per la salute umana ed animale. Al fine di garantire programmi di monitoraggio affidabili per una corretta valutazione del rischio associato alla loro esposizione e il rispetto dei livelli massimi ammissibili stabiliti in varie derrate alimentari, sono necessari metodi validati con caratteristiche che soddisfino determinati criteri di accettabilità. In questo articolo viene presentata una breve panoramica di recenti attività svolte presso l'Istituto di Scienze delle Produzioni Alimentari del Consiglio Nazionale delle Ricerche (ISPA-CNR) per lo sviluppo e validazione di metodi analitici per la determinazione di micotossine nei cereali e prodotti a base di cereali.

Introduction LC-MS/(MS) is routinely used for the simultaneous determination of mycotoxins in food and feed although official methods using this technique have not yet been adopted by CEN and AOAC International. Objective Conduct a proficiency test (PT) for the simultaneous determination of up to 11 mycotoxins [aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), ochratoxin A (OTA), deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEA), fumonisin B1 (FB1) and fumonisin B2 (FB2)] in maize using LC-MS/(MS) to benchmark laboratories currently using this technique and to obtain information on currently used methodologies and method-related performances. Methods Each participant received the following: instructions; a comprehensive questionnaire; a mixed mycotoxins calibration solution; a spiking solution (AFB1, AFB2, AFG1 and AFG2, OTA, DON, T-2, HT-2, ZEA, FB1 and FB2); two test materials, namely a contaminated maize sample and a blank maize sample to be spiked with a spiking solution containing 11 mycotoxins. Laboratory results were rated with z-scores. Results Of the 64 laboratories enrolled in the PT, 41 laboratories from 14 countries returned 43 sets of results for various combinations of analytes. The majority of laboratories (61%) reported results for all 11 mycotoxins whereas the remaining laboratories reported results for a restricted combination (from 2 to 10 analytes). For contaminated maize and spiked maize the percentage of satisfactory z-score values (z < 2) were: DON 55% and 49%, FB1 50% and 30%, FB2 52% and 38%, ZEA 68% and 64%, T-2+HT-2 toxins 82% and 85%, OTA 58% and 60%, AFB1 56% and 62%, AFG1 73% and 84%, AFB2 40% and 78%, AFG2 64% and 78%. The poorest performance (z > 3) was obtained for FB1 (31%), FB2 (32%), AFB1 (32%) and AFB2 (32%) in contaminated maize and for DON (35%), FB1 (63%) and FB2 (52%) in spiked maize. Mean recovery results were acceptable for all mycotoxins (74% to 109%), except for fumonisins, where these were unacceptably high (159% for FB1 and 163% for FB2). Conclusion A robust and reliable method for simultaneous determination of 11 mycotoxins in maize could not be identified from the results of this PT. Additional experimental work is necessary to set up a method suitable for interlaboratory validation. The results of this PT and the relevant method's details can be useful to identify methodology strengths and weaknesses.

The recent Commission Recommendation 2013/165/EU, asked for collection of more data on T-2 and HT-2 toxins occurrence in cereals and cereal products and emphasized that "in case the used method of analysis enables it, it would be appropriate to analyze also the masked mycotoxins, in particular the mono- and di-glycosylated conjugates of -T2 and HT-2 toxin". Addressing these issues, the aim of this work was to obtain more comprehensive information on the co-occurrence of T-2 and HT-2 toxins and their glucosylated derivatives in naturally contaminated cereal samples. For these purposes, barley samples originating from a Northern Italian area, were analyzed by LC-HRMS for the presence of T-2, HT-2, and relevant glucosyl derivatives. Quantitative analysis of T-2 and HT-2 glucosides was performed for the first time using a recently made available standard of T-2 glucoside. A widespread co-occurrence of the glucosyl derivative of HT-2 with the unconjugated toxin was observed in unprocessed barley grains, whereas the monoglucosyl derivative of T-2 toxin was detected in only a few samples and at low µg/kg levels. The ratio between glucosylated toxins (sum of T-2 and HT-2 glucosides) and native toxins (sum of T-2 and HT-2) ranged from 2 to 283%. Moreover, taking advantage of the possibility of retrospective analysis of full scan HRMS chromatograms, samples were also screened for the presence of other type-A trichothecenes, namely neosolaniol, diacetoxyscirpenol and their mono-glucosyl derivatives, which were detected at trace levels. A subset of nine different samples was subjected to micro-maltation in order to carry out a preliminary investigation on the fate of T-2, HT-2 and relevant glucosides along the malting process. Mycotoxin reduction from cleaned barley to malt was observed at rates ranging from 4% to 87%.

This paper reports a new method for the determination of T-2 and HT-2 toxins and their glucosylated derivatives in cereals, and some survey data aimed at obtaining more comprehensive information on the co-occurrence of T-2 and HT-2 toxins and their glucosylated derivatives in naturally contaminated cereal samples. For these purposes, barley samples originating from a Northern Italian area were analysed by LC-HRMS for the presence of T-2, HT-2 and relevant glucosyl derivatives. Quantitative analysis of T-2 and HT-2 glucosides was performed for the first time using a recently made available standard of T-2 glucoside. The glucosyl derivative of HT-2 was detected at levels up to 163 mu gkg(-1) in 17 of the 18 analysed unprocessed barley grains, whereas the monoglucosyl derivative of T-2 toxin was detected in only a few samples and at low mu gkg(-1) levels. The ratio between glucosylated toxins (sum of T-2 and HT-2 glucosides) and native toxins (sum of T-2 and HT-2) ranged from 2% to 283%. Moreover, taking advantage of the possibility of retrospective analysis of full-scan HRMS chromatograms, samples were also screened for the presence of other type-A trichothecenes, namely neosolaniol, diacetoxyscirpenol and their monoglucosyl derivatives, which were detected at trace levels. A subset of nine different samples was subjected to micro-maltation in order to carry out a preliminary investigation on the fate of T-2, HT-2 and relevant glucosides along the malting process. Mycotoxin reduction from cleaned barley to malt was observed at rates ranging from 4% to 87%.

Soybean and soy meal samples collected during the harvest season 2008-2009 in the soybean-growing area of Córdoba Province in Argentina were analysed for T-2 and HT-2 toxins occurrence. These mycotoxins were detected using HPLC analysis with fluorescence detection after derivatisation with 1-anthronylnitrile and immunoaffinity column clean-up. Characteristics of in-house validated method such as accuracy, precision, detection and quantification limits were defined by means of recovery test with spiked soybean and soy meal samples. Mean recoveries for T-2 within the spiking range 125-500 ?g/kg, were 90.9 and 81.3% for soybean and soy meal, respectively with a within-laboratory relative standard deviation <10%. Analysis of samples spiked with HT-2 in the same range gave a mean recovery of 70.2 and 77.5% for soybean and soy meal, respectively, with relative standard deviations <12%. The limit of detection for the method was 25 ?g/kg for T-2 and HT-2, based on a signal-to-noise ratio 3:1 and the limit of quantification was established as three times the detection limit. Out of 64 samples, only two soybean samples showed contamination with A-type trichothecenes evaluated. Confirmatory analyses of the contaminated samples were performed by LC-MS/MS. This study demonstrated low incidences and levels of T-2 and HT-2 in soybean harvested among the areas in the Cordoba Province.

Screening methods are defined as methods that are used to detect the presence of a substance or class of substances at the level of interest. These methods must have the capability of high sample throughput when being used to screen large numbers of samples for potential noncompliant results. Before using a screening method for practical applications, its fitness for the intended purpose needs to be demonstrated. This is normally achieved by conducting a validation study, comparing method performance against predefined criteria. Official guidelines recently established by the European Union for the evaluation of fitnessfor-purpose performance parameters of screening methods to be used for the detection of mycotoxins in foods are presented and discussed herein. Practical applications of this evaluation scheme for single-and interlaboratory validation studies, as well as relevant information on screening method performances are reviewed, with emphasis on the impact of mycotoxin contamination in real samples on the fitness-for-purpose of the screening test. Lastly, validation follow-up is discussed in terms of extension of the scope of the method (increasing the range of application in terms of mycotoxin/ matrix combinations), method implementation and verification, and evaluation of the method's applicability to modified mycotoxins.

The synthesis of partially hydrolyzed fumonisins (PHFB1 and PHFB2) and hydrolyzed fumonisins (HFB1 and HFB2) by chemicalhydrolysis of pure fumonisins (FB1 and FB2) is reported together with the isolation and characterization by liquidchromatography-high-resolution mass spectrometry (LC-HRMS). Two structural isomers of partially hydrolyzed forms ofFB1 and FB2 were identified, namely PHFB1a and PHFB1b and PHFB2a and PHFB2b. Reaction yields were 21% for PHFB1(sum of the two isomers), 52% for HFB1, 31% for PHFB2 (sum of the two isomers) and 30% for HFB2. Purity of each isolatedcompound was >98%.An LC-HRMS method for the simultaneous determination of fumonisins and their partially and totally hydrolyzedderivatives was applied to 24 naturally contaminated samples of maize and maize-based products. The majority of samples(18 out of 24) were contaminated with fumonisins B1 and B2. Fumonisins co-occurred with both partially hydrolyzed andhydrolyzed fumonisins in four nixtamalized samples (three masa flours and one tortilla chips). Co-occurrence of fumonisins withpartially hydrolyzed fumonisins was also recorded in one sample ofmaize kernels and four samples ofmaize-based products (i.e.maize meal, cous-cous, corn-cakes and cornflakes). Mycotoxins levels ranged from 60 to 5700 ?g/kg for fumonisins (sum of FB1and FB2), from 10 to 210 ?g/kg for partially hydrolyzed fumonisins (sum of PHFB1 and PHFB2) and from 30 to 200 ?g/kg forhydrolyzed fumonisins (sum of HFB1 and HFB2). This is the first report of the isolation of PHFB2 and the co-occurrence of FB1,FB2, PHFB1, PHFB2, HFB1 and HFB2 in maize products. Considering the growing use of nixtamalized and maize-based products,the monitoring of fumonisins and their partially and totally hydrolyzed forms in these products may represent an importantcontributing factor in evaluating the relevant human risk exposure.

The analytical performances of a lateral flow immunoassay (AFLA-V AQUA(TM), Vicam a Waters Business) forthe determination of aflatoxins in maize were evaluated according to Commission Regulation (EU) No. 519/2014. This case study showed how a critical evaluation of validation results can provide a quite informativepicture of kit performances for its use for different purposes. The validation study, performed at aflatoxin B1levels encompassing EU maximum permitted levels (up to 8 ug kg-1), provided information on the methodprecision profile, cut off values, and false suspect and false negative rates. The total precision expressed asrelative standard deviation varied from 14 to 29% for contaminated samples. Cut off values calculatedconsidering 2 ug kg-1 or 4 mg kg-1 as the screening target concentration were 1.24 and 2.18 ug kg-1,respectively, whereas the resultant false suspect rate for blanks was 42% and 8%, respectively. The falsenegative rate for samples containing the analyte at higher concentrations was found to be lower than1%. Analysis of naturally contaminated maize samples performed by the lateral flow immunoassay andthe AOAC official method revealed a good correlation (parameters of the linear regression: r = 0.97,slope = 0.96) between the obtained results. A satisfactory agreement between reference values and testresults was also obtained in the analysis of reference materials. Finally, a QC protocol based onguidelines suggested in the EU regulation was designed to verify kit performances over time and in newproduction lots.

Recent advances in establishing harmonized guidelines for validation of screening methods are reviewed. Emphasis is given to guidelines set in the Regulation 519/2014/EU that specifies validation criteria for mycotoxin screening methods to be used for official control purposes. The application of this validation scheme is demonstrated through a case study. A commercial lateral flow immunoassay for deoxynivalenol has been and evaluated for its applicability to verify wheat compliance with EC maximum permitted level. The validation design provided information on the precision profile of the method, cut-off, false suspect and false negative rates of samples containing deoxynivalenol above the legal limit. The influence of the co-occurrence of major deoxynivalenol modified forms, i.e. 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, and deoxynivalenol-3-glucoside on test results, was evaluated by a factorial design. Finally, the applicability of the validated immunoassay was demonstrated by analysis of naturally contaminated wheat samples, and comparison with results obtained by a LC-MS/MS confirmatory method.

Tra le metodiche di screening da utilizzare on site, quelle basate sull'uso di dipstick o lateral flow devices sono attualmente le più diffuse per la determinazione rapida delle micotossine sottoposte a regolamentazione in cereali non processati. Prima del loro impiego, tuttavia, anche i metodi rapidi, come quelli convenzionali, devono essere sottoposti a esperimenti di validazione, al fine di valutare le loro caratteristiche analitiche. Le linee guida per la validazione dei metodi di screening per micotossine sono descritte nel recente Regolamento 519/2014/EU.Obiettivo del presente lavoro è stato quello di elaborare un disegno sperimentale per la validazione intra-laboratorio di un metodo rapido per la determinazione semi-quantitativa del deossinivalenolo (DON) in frumento duro, in accordo con le linee guida del suddetto Regolamento. Il metodo è basato su un test immunocromatografico a flusso laterale. Il disegno sperimentale applicato ha fornito informazioni sul profilo di precisione del metodo, sul cut-off e sulle percentuali di falsi positivi e falsi negativi. La precisione intermedia (riproducibilità intra-laboratorio) variava tra 16 e 28%. L'analisi della varianza (ANOVA) ha inoltre rivelato che, tra le varie sorgenti di errore considerate (variazione tra giorni, varietà di frumento, differenti lotti di produzione e ripetibilità) il maggiore contributo è rappresentato dalla ripetibilità. La percentuale di falsi positivi per campioni contaminati al di sotto del limite di legge è risultata inferiore all'1%, mentre la percentuale di falsi negativi per campioni contaminati al di sopra del limite di legge era 1.7%. Inoltre, è stato elaborato un disegno fattoriale per valutare quantitativamente l'influenza delle principali forme modificate del DON (3-acetil-DON, 15-acetil-DON e DON-3-glucoside) sui risultati del test, e in particolare sul rischio di falsi positivi. Infine, l'applicabilità del metodo validato a campioni reali è stata valutata mediante analisi di 47 campioni di frumento duro naturalmente contaminati. I risultati delle analisi hanno mostrato una buona correlazione (r = 0.87) con quelli ottenuti mediante un metodo di conferma LC-MS/MS.

Le fumonisine B1 (FB1) e B2 (FB2) sono micotossine prodotte principalmente dai funghi del genere Fusarium, frequenti contaminanti del mais. Per il suo valore nutritivo e per le svariate utilizzazioni dei suoi prodotti e sottoprodotti, il mais rappresenta una componente importante dell'alimentazione umana. In alcuni paesi del Sud America e nel Messico il mais viene consumato previa cottura in presenza di una soluzione di idrossido di calcio. Questo processo, noto come nixtamalizzazione, produce un impasto chiamato "masa" che possiede proprietà nutrizionali superiori a quelle del mais di partenza. La "masa" viene poi impiegata per la produzione di svariati prodotti derivati come tortillas, tamales e arepas. Durante il processo di nixtamalizzazione si verifica la rimozione di uno o di entrambi i residui di acido tricarballilico delle fumonisine dando origine rispettivamente alle fumonisine parzialmente idrolizzate (PHFBs) o alle fumonisine idrolizzate (HFBs). Recentemente presso i laboratori ISPA-CNR sono stati prodotti, isolati e caratterizzati per la prima volta standard puri (purezza >98%) di PHFB1, PHFB2, HFB1 e HFB2. La disponibilità di tali standard ha permesso di valutare l'effetto del processo di nixtamalizzazione (condotta su scala da laboratorio) sul contenuto delle fumonisine e delle relative forme idrolizzate. Il mais di partenza, le frazioni intermedie (acque di steeping e di lavaggio) e la "masa" finale sono state analizzate mediante LC-HRMS per determinarne il contenuto di FBs, PHFBs e HFBs. Sono stati condotti anche esperimenti di controllo che prevedevano la cottura del mais in assenza di idrossido di calcio. I risultati hanno evidenziato che la nixtamalizzazione riduceva il contenuto di FBs e PHFBs nella masa finale, mentre favoriva la produzione di HFBs. Tuttavia, il contenuto complessivo delle tre forme di fumonisine nelle frazioni raccolte risultava ben superiore al 100% (fino al 180%) indicando che la nixtamalizzazione rendeva disponibili forme di fumonisine complessate con i componenti della matrice che venivano a loro volta convertite nelle relative forme idrolizzate. Al contrario, la cottura del mais in assenza di idrossido di calcio non rilasciava HFBs e quasi il 100% di fumonisine iniziali veniva raccolto nelle frazioni intermedie e nella mais cotto. Considerata la minore tossicità delle forme idrolizzate delle fumonisine rispetto alle forme native si può affermare che la nixtamalizzazione produce alimenti più salubri rispetto al mais di partenza.