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Seventy-one (71) food samples were analyzed for the mycotoxin fumonisin by a monoclonal antibody based competitive enzyme-linked immunosorbent assay (ELISA). Fumonisins were detected primarily in corn-based products with 7/12, 2/2 and 1/3 and 1/7 yellow cornmeal, blue cornmeal, corn muffin mix, and mixed grain cereal samples yielding positive results, respectively. When the positive samples and randomly selected negative samples were assessed by other methods, correlations (r values) between ELISA and gas chromatography-mass spectrometry (GC-MS), ELISA and high-pressure liquid chromatography (HPLC) and GC-MS and HPLC were 0.478 (p0.05), 0.512 (p0.05), and 0.946 (p0.01), respectively. The results suggested that although the immunoassay could be used for screening of fumonisin in food samples, higher estimates were attained by ELISA than by the other two methods particularly in the more contaminated samples. These observations may result from differences in sample preparation among the methods or because of the presence of structurally related compounds in extracts that are detectable by ELISA but not the other two methods.

Ingestion of food is considered a major route of exposure to many contaminants including mycotoxins. The amount of mycotoxin resisting to the digestion process and potentially absorbable by the systemic circulation is only a smaller part of that ingested. In vitro digestion models turn useful for evaluating mycotoxins bioaccessibility during the intestinal transit and can be intended as a valuable tool for the assessment of mycotoxin bioavailability in food. In this paper we describe a study aimed at investigating toxicity of in vitro gastro-duodenal digests of mycotoxin contaminated bread collected along the digestion time-course. Toxicity tests were carried out on a sensitive RPMI lymphoid B cell line chosen as the most suitable lineage to assess toxicity retained by gastro-duodenal digests. In parallel, a chemical quantification of T-2 and HT-2 toxins contaminating the bread digests was accomplished during the gastric and duodenal transit. The digestive fluids undergoing chemical and toxicological analysis were collected at the beginning and end of gastric phase, and after completion of the duodenal phase. Results proved that a correlation between HT-2 content and toxicity did exist although a more persistent toxic activity was displayed in the later stage of the duodenal phase. This persistent toxicity might be explained by the co-occurrence of unknown HT-2-related conjugates or metabolites formed during digestion.

An experimental protocol for evaluating performances of qualitative methods through interlaboratory validation has been designed and applied to a multiplex dipstick kit for the determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol, and fumonisins B1 and B2 in wheat and maize. The test is intended for screening of cereals for the presence/absence of these mycotoxins at maximum permitted levels established by European legislation. The response of the measurement is determined with a reader device. Decision on whether a sample is classified as positive or negative is taken by comparing the measured response with a cut off value which has been established based on the results from the method validation. The collaborative study involved 12 laboratories and included a training phase enabling the participants to familiarise with the protocol. The interlaboratory validation design consisted of three steps, namely (1) estimating the precision under reproducibility conditio...

T-2 and HT-2 toxins are Fusarium mycotoxins frequently occurring in cereal-based products with a toxic effect ascertained on different biological systems. Despite other mycotoxins, to date bioaccessibility of T-2 and HT-2 toxins has never been investigated. In order to provide insights on T-2 and HT-2 stability and bioaccessibility along digestion in the upper intestinal tract, differently contaminated bread samples produced at laboratory scale were submitted to in vitro digestion experiments. Two different contaminated bread models were prepared with this aim. One employed naturally contaminated wheat flour for production of naturally contaminated bread, whereas in the other model dough was fortified with both mycotoxins during bread preparation before undergoing the leavening and baking process. Gastro-duodenal fluids were collected at different time-points along simulated digestion and mycotoxin content determined by LC-High Resolution–Mass Spectrometry. Our data report that HT-2 content in the beginning of the gastric phase was not significantly different from what was recorded at the end of the duodenal phase, although an apparent decrease of the signal was displayed in the early duodenal phase in both bread models tested. By contrast, the consistent drop of approximately 50% observed for T-2 when the duodenal digestion started remained constant until the digestion reached completion. In conclusion both approaches provided similar results thus considering incurred food models valid alternatives to undertake such investigations. Finally, bioaccessibility of T2 and HT2 was calculated in both food models tested with a higher bioaccessibility found in artificially contaminated bread samples. Industrial Relevance: Food is very complex both in composition and structure; therefore, generic realistic models that can mimic this complexity are required. Such models would greatly facilitate evaluation of the impact of changing composition or processing conditions on nutrition and safety. The primary scope of this work (part of the EU funded DREAM project) is to integrate experimental and mathematical approaches elsewhere optimised to develop a cereal-based food model realistic enough to be used by the industry and sufficiently versatile to be used as predictive tool of food behaviour. This is would be also applicable to study the release of contaminants from food such as mycotoxins. In general, food parameters may affect mycotoxins bioavailability from foods. A number of mycotoxins are currently regulated in foodstuffs by the European Commission and as a consequence there is growing interest and concern of the public health authorities for the presence of mycotoxins in human food. Due to the different chemical structure and stability, the fate of mycotoxins during food processing and the relevant bioaccessibility upon digestion process vary considerably depending on the different food preparations. Limited data are currently available on the fate of trichothecenes during food processing and human digestion. Therefore, the fate of these mycotoxins during processing of some specific food models is of paramount importance to be assessed. The bioaccessibility of trichothecenes (deoxynivalenol, T2 and HT-2 toxins) from a selected cereal based food model evaluated by an in vitro digestion model is also important to understand the safety of the final food.

The synthesis of partially hydrolyzed fumonisins (PHFB1 and PHFB2) and hydrolyzed fumonisins (HFB1 and HFB2) by chemical hydrolysis of pure fumonisins (FB1 and FB2) is reported together with the isolation and characterization by liquid chromatography–high-resolution mass spectrometry (LC–HRMS). Two structural isomers of partially hydrolyzed forms of FB1 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 isolated compound was >98%. An LC–HRMS method for the simultaneous determination of fumonisins and their partially and totally hydrolyzed derivatives 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 and hydrolyzed fumonisins in four nixtamalized samples (three masa flours and one tortilla chips). Co-occurrence of fumonisins with partially hydrolyzed fumonisins was also recorded in one sample of maize kernels and four samples of maize-based products (i.e. maize meal, cous-cous, corn-cakes and cornflakes). Mycotoxins levels ranged from 60 to 5700 µg/kg for fumonisins (sum of FB1 and FB2), from 10 to 210 µg/kg for partially hydrolyzed fumonisins (sum of PHFB1 and PHFB2) and from 30 to 200 µg/kg for hydrolyzed 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 important contributing factor in evaluating the relevant human risk exposure. Copyright © 2014 John Wiley & Sons, Ltd.

Subsistence farmers are exposed to a range of mycotoxins. This study applied novel urinary multi-mycotoxin LC-MS/MS methods to determine multiple exposure biomarkers in the high oesophageal cancer region, Transkei, South Africa. Fifty-three female participants donated part of their maize-based evening meal and first void morning urine, which was analysed both with sample clean-up (single and multi-biomarker) and by a 'dilute-and-shoot' multi-biomarker method. Results were corrected for recovery with LOD for not detected. A single biomarker method detected fumonisin B1 (FB1) (87% incidence; mean ± standard deviation 0.342 ± 0.466 ng/mg creatinine) and deoxynivalenol (100%; mean 20.4 ± 49.4 ng/mg creatinine) after hydrolysis with b-glucuronidase. The multi-biomarker 'dilute-and-shoot' method indicated deoxynivalenol-15-glucuronide was predominantly present. A multi-biomarker method with b-glucuronidase and immunoaffinity clean-up determined zearalenone (100%; 0.529 ± 1.60 ng/mg creatinine), FB1 (96%; 1.52 ± 2.17 ng/mg creatinine), a-zearalenol (92%; 0.614 ± 1.91 ng/mg creatinine), deoxynivalenol (87%; 11.3 ± 27.1 ng/mg creatinine), b-zearalenol (75%; 0.702 ± 2.95 ng/mg creatinine) and ochratoxin A (98%; 0.041 ± 0.086 ng/mg creatinine). These demonstrate the value of multi-biomarker methods in measuring exposures in populations exposed to multiple mycotoxins. This is the first finding of urinary deoxynivalenol, zearalenone, their conjugates, ochratoxin A and zearalenols in Transkei.

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 represent a 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 mass spectrometry (LC-MS/MS) was used for the identification and chemical characterization of these metabolites. F. langsethiae isolates 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 one mono-glucoside derivative of DAS were identified and characterized. The screening for the presence/absence of glucosylated trichothecenes in analyzed fungal extracts revealed a general co-occurrence of these derivatives with the parent toxin at levels that could be roughly estimated to account up to 37% of the relevant unconjugated toxin. This is the first report of the production of glucosylated trichothecenes by F. langsethiae cultured on small grains.

The results of a proficiency test for the LC-MS/(MS) determination of up to 11 mycotoxins (aflatoxins B1, B2, G1 and G2, fumonisins B1 and B2, 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.

Maize harvested in the Centane region of the former Transkei, Eastern Cape Province, South Africa, by subsistence farmers has been shown over many seasons to be contaminated with fumonisin mycotoxins. However, there are limited data on the presence of other mycotoxins. Two multimycotoxin LC-MS/MS methods were applied to good and moldy maize samples, as separated by the farmers themselves from the 2011 harvest. One method involved extract cleanup on multitoxin immunoaffinity columns before LC-MS/MS analysis for aflatoxins, fumonisins, deoxynivalenol (DON), zearalenone (ZEN), and T-2 and HT-2 toxins. The other method was based on a "dilute-and-shoot" approach for the above mycotoxins and a wide range of other fungal secondary metabolites. Both methods showed high incidences of fumonisins B1 and B2 (FB1 and FB2) in good maize (100% for both by the first method, means were 2083 and 927 μg/kg for the two analogues; 93% for both by the second method, positive means of 2764 and 1050 μg/kg, respectively). All samples of moldy maize were contaminated (mean FB1 of 27.64 and 35.98 mg/kg, respectively; mean FB2 of 10.58 and 14.14 mg/kg, respectively). Comparison of the two methods for FB1 and FB2 over the entire range of samples gave R(2) values 0.9144 and 0.8859, respectively. Low levels of DON were found by both methods (positive means of 12 and 4.7 μg/kg in good maize, respectively, and of 14 and 5.8 μg/kg in moldy maize, respectively). ZEN was determined with positive means of 108 and 25 μg/kg in good maize, respectively, and of 111 and 135 μg/kg in moldy maize, respectively. No aflatoxins, OTA, or T-2 or HT-2 toxins were detected. A wide range of other Fusarium , Aspergillus , Alternaria , and Penicillium mycotoxins and secondary metabolites were determined.

The multi-biomarker approach was used to validate urinary biomarkers in piglets administered boluses contaminated with mixtures of deoxynivalenol (DON), aflatoxin B1 (AFB1), fumonisin B1 (FB1), zearalenone (ZEA) and ochratoxin A (OTA) at different concentrations. Boluses contaminated with mycotoxins were prepared by slurrying and freezedrying feed material fortified with culture extracts of selected toxigenic fungi. Piglets were individually placed in metabolic cages to collect urine before gavage and 24 h post dose. Urine samples were hydrolysed with β-glucuronidase and analysed by a multi-biomarker LC-MS/MS method developed and validated to identify and measure biomarkers of FB1, OTA, DON, ZEA and AFB1. Urinary levels of FB1, OTA, DON + de-epoxy-deoxynivalenol, ZEA + alphazearalenol and aflatoxin M1 were selected as biomarkers of FB1, OTA, DON, ZEA and AFB1, respectively. Mean percentages of dietary mycotoxins excreted as biomarkers in 24 h post dose urine were 36.8% for ZEA, 28.5% for DON, 2.6% FB1, 2.6% for OTA and 2.5% for AFB1. A good correlation was observed between the amount of mycotoxins ingested and the amount of relevant biomarkers excreted in 24 h post dose urine. Linear dose-response correlation coefficients ranged between 0.68 and 0.78 for the tested couples of mycotoxin/biomarker. The good sensitivity of the LC-MS/MS method and the good dose-response correlations observed in this study permitted to validate the selected mycotoxin biomarkers in piglets at dietary levels close to the maximum permitted levels reported in Commission Directive 2003/100/EC for AFB1 and the guidance values reported in Commission Recommendation 2006/576/EC for DON, ZEA, OTA and FB1.

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. Copyright © 2012 John Wiley & Sons, Ltd.

A sensitive and accurate method employing a single stage high resolution mass spectrometer equipped with a high-energy collision-dissociation cell (HCD) for the simultaneous determination of deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in a processed bread model food has been developed. Two sample pre-treatment routes for the extraction of these mycotoxins were investigated, based on Mycosep(®) column clean up or QuEChERS-like procedure, respectively. The former approach suffered less from matrix effects and allowed to achieve in bread samples LODs of 7, 12 and 17 ng/g for T-2, HT-2 and DON, respectively, with 0.5 ppm mass accuracy. Two acquisition modes, full scan MS and all ion fragmentation, exploiting the fragmentation features offered by an HCD chamber and integrated within the Orbitrap analyser, were compared for quantitative purposes. The method was applied to investigate the degradation of these mycotoxins during bread processing using a bread model food. Most T-2 hydrolyzed to HT-2 during dough preparation, and about 20-30% of HT-2 and DON was degraded during bread baking.

International audience; A liquid chromatography - high resolution mass spectrometry (LC-HRMS) method for the simultaneous determination of aflatoxins (B1, B2, G1, G2), ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in wheat flour, barley flour and crisp bread has been developed. Data presented show the process of obtaining. Mycotoxin fragmentation patterns obtained by High Energy Collision Dissociation (HCD) have been 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 allowed to monitor the pseudo-molecular ion and an additional characteristic fragment (for each mycotoxin) with mass accuracy in the range 0.1 - 3.9 ppm, meeting the current European regulatory requirements for LC-MS confirmatory analysis. A sample preparation procedure based on polymeric solid phase extraction cartridges was applied, allowing recoveries higher than 74% for all tested toxins, with relative standard deviation lower than 13%. Detection limits in the range 0.5 - 3.4 µg/kg were obtained for all tested matrices. A critical comparison between the proposed method and a validated method based on triple quadrupole mass spectrometry showed similar performances in terms of detection limits, recoveries and repeatability, and matrix effects. Based on an efficient sample extraction and clean up, the LC-HCD-HRMS method developed herein represents a reliable and robust alternative tool for mycotoxin analysis in food matrices as compared to well established triple quadrupole based approaches.

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.

Humans and animals can be simultaneously exposed through the diet to different mycotoxins, including aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, and fumonisins, which are the most important. Evaluation of the frequency and levels of human and animal exposure to these mycotoxins can be performed by measuring the levels of the relevant biomarkers in urine. Available data on the toxicokinetics of these mycotoxins in animals suggest that aflatoxin M(1) (AFM(1)), ochratoxin A (OTA), deoxynivalenol (DON)/de-epoxydeoxynivalenol (DOM-1), alpha-zearalenol (α-ZOL)/beta-zearalenol (β-ZOL), and fumonisin B(1) (FB(1)) can be used as urinary biomarkers. A liquid chromatographic-tandem mass spectrometric method has been developed for simultaneous determination of these mycotoxin biomarkers in human or animal urine. Urine samples were purified and concentrated by a double cleanup approach, using a multitoxin immunoaffinity column and a reversed-phase SPE Oasis HLB column. Separation of the biomarkers was performed by reversed-phase chromatography using a multi-step linear methanol-water gradient containing 0.5% acetic acid as mobile phase. Detection and quantification of the biomarkers were performed by triple quadrupole mass spectrometry (LC-ESI-MS/MS). The clean-up conditions were optimised to obtain maximum analyte recovery and high sensitivity. Recovery from spiked samples was performed at four levels in the range 0.03-12 ng mL(-1), using matrix-matched calibration curves for quantification. Mean recoveries of the biomarkers tested ranged from 62 to 96% with relative standard deviations of 3-20%. Enzymatic digestion with β-glucuronidase/sulfatase resulted in increased concentrations of the biomarkers, in both human and pig urine, in most samples containing measurable concentrations of DON, DOM-1, OTA, α-ZOL, or β-ZOL. A highly variable increase was observed between individuals. Co-occurrence of OTA and DON in human urine is reported herein for the first time.

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.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm motility by CASA system. Fumonisin B1 did not affect viability of fresh spermatozoa after 2 h exposure up to 25 μM. Damage on sperm chromatin structure was observed only in one frozen sample after exposure up to 2.5 × 10 −5 μM FB1 without associated increase of ROS. Increase of ROS, at FB1 levels up to 2.5 × 10 −4 μM, was found on another frozen-thawed sperm sample, may be as a consequence of seminal plasma removal. At 7.5 and 15 μM, FB1 induced reduction of total and progressive motility.

Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B(1). Six unique sequences were obtained, each showing improved binding to fumonisin B(1) compared to controls. Sequence FB(1) 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.

The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate the toxic effect of mycotoxin on their chromatin structure by flow cytometry. Different parameters of the DNA fragmentation index (DFI), such as the mean ( X ¯ - DFI ), the percentage (%-DFI), and the standard deviation (SD-DFI), were analyzed. Urine samples showed a mean level of 32.3 ng/mL ZEA with significantly higher concentrations in northeastern European samples than in Italian samples, probably in relation to climatic and feeding differences. The toxic effects of ZEA-containing urine samples on SCSA parameters were found at low ZEA concentrations and were mainly observed in Italian samples. By using mycotoxin standards, ZEA, α-zearalenol, and β-zearalenol proved to be more toxic compounds for sperm chromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized.

Fusarium head blight (FHB) is one of the major diseases of wheat (both common and durum wheat) caused by various fungi including Microdochium nivale and different Fusarium species. Most of the Fusarium species associated with FHB (mainly F. graminearum, F. culmorum and F. sporotrichioides), under favourable environmental conditions, can produce various toxic secondary metabolites (mycotoxins) that can contaminate grains. The major Fusarium mycotoxins that can occur in wheat and derived products are deoxynivalenol, nivalenol, T-2 and HT-2 toxins, and zearalenone. Processing has generally significant effects on the levels of mycotoxins in the final products. Deoxynivalenol is typically concentrated in the bran coat which is removed in the production of semolina; consequently, a consistent reduction of deoxynivalenol levels has been observed during each of the processing steps, from raw durum wheat to pasta production. To allow monitoring programs and protect consumers' health, several analytical me...

Ochratoxin A contamination of red wines might be quite severe in certain high-risk regions and vintages, thus requiring corrective measures to fulfill acceptable standards for human consumption. This work proposes an innovative and environmentally friendly corrective measure to reduce ochratoxin A levels by repassage of contaminated musts or wines over grape pomaces having no or little ochratoxin A contamination. Grape pomaces have a high affinity for ochratoxin A and have been shown to remove ochratoxin A from must and wine during vinification. Time course experiments showed that ochratoxin A adsorption by pomaces is a rapid process, reaching equilibrium in less than 10 h, and is not affected by the tested toxin concentrations. Repassage of wine from Primitivo grapes spiked with 2-10 microg/kg ochratoxin A over pomaces obtained from the same grapes removed up to 65% ochratoxin A within 24 h. Similar results (50-65% ochratoxin A reduction) were obtained with Primitivo or Negroamaro wines repassed over pomaces from different grape varieties including white grapes (Malvasia, Greco di Tufo) and red grapes (Sangiovese, Aglianico). Grape pomaces maintained a good efficacy in removing ochratoxin A after being reused four times. Unlike other enological fining agents, the use of grape pomaces to adsorb ochratoxin A from red wines of the same grape variety (Primitivo) did not affect relevant wine quality parameters, including color intensity and health-promoting phenolic content (trans-resveratrol, quercetin, total polyphenols). These quality parameters were instead positively or negatively affected when contaminated wines were repassed over grape pomaces from other grape varieties, the effect being related to the intrinsic characteristics of the pomace variety. The proposed decontamination procedure can be applied in a modern winery provided that contaminated grapes are identified early and processed separately from uncontaminated grapes.

We review recent advances in analytical methods for trichothecene mycotoxins in cereals and cereal-based food products with emphasis on their advantages and limitations. We describe several traditional methods based on chromatographic or immunochemical techniques, which allow sensitive, accurate analysis or rapid screening of major trichothecenes throughout the cereal-food chain. In addition, we discuss novel technologies for trichothecene analysis, including tandem mass spectrometry, fluorescence-polarization immunoassays, lateral flow devices, infrared spectroscopy and optical biosensors.

Aspergillus niger has been recently found to produce fumonisin B(2) (FB(2)). Thirty-one strains belonging to four Aspergillus species isolated from grape were evaluated for FB(2) production on agar plates. Four out of eight strains of A. niger produced FB(2) (29-293 microg g(-1)). None of the strains of A. uvarum (n = 7), A. tubingensis (8) and A. carbonarius (8) produced detectable amounts of toxin. The capability to produce FB(2) was also confirmed by some A. niger strains artificially inoculated on grape berries. Natural occurrence of FB(2), at levels of 0.01 and 0.4 microg ml(-1), was found in two samples of must collected in Apulian cellars in 2007. This is the first report of FB(2) contamination in must. These findings suggest that there is a potential risk of exposure to FB(2) in the grape-wine chain for consumers and that A. niger may represent the major fumonisin-producing species among black Aspergilli occurring on grapes.

In 2004, the European Commission approved the specific support action 'Integration of Mycotoxin and Toxigenic Fungi Research for Food Safety in the Global System' (MycoGlobe, contract FOOD-CT-2004-007174) within the Sixth Framework Programme, Food Quality and Safety. The aim of the MycoGlobe project (http://mycoglobe.ispa.cnr.it) was to implement the outcomes of a wide range of European research projects in the area of mycotoxins and toxigenic fungi by supporting, stimulating and facilitating cooperation between countries in the European Union and other countries that have bilateral scientific and technological cooperation agreement with the European Union (such as USA, Australia and South America). Through a series of conferences and interactions between scientists worldwide, MycoGlobe was a very successful project. The scientific significance of the MycoGlobe project consisted in the spread of knowledge of advanced research tools in genomics and sophisticated and rapid detection systems for mycotoxins and toxigenic fungi; and evaluation of research policy and procedures to achieve best practice for enhancement of food quality and safety by elimination of mycotoxins and toxigenic fungi from commodities. The socio-economic significance of the project was the setting up of a global collaborative network for research and technology transfer in the field of mycotoxins and toxigenic fungi, particularly for the benefit of the developing countries. A relevant outcome of the project was also the launching of the International Society for Mycotoxicology (http://www.mycotoxsociety. org) to promote research on mycotoxins and toxigenic fungi, thereby leading to prevention and reduction in exposure to mycotoxins, enhanced food safety and a greater public awareness of this area.

Cyclodextrins, cyclic oligosaccharides composed of amylose subunits, are known to interact with mycotoxins. The interactions may be useful to analytical chemists by altering the properties of the mycotoxin of interest, namely the chromatographic properties, electrophoretic properties, fluorescence, or absorption of these fungal metabolites. Practical applications of these effects have been the incorporation of cyclodextrins into high-performance liquid chromatography and capillary electrophoresis methods for mycotoxin detection. Specific mycotoxins include those with a native fluorescence such as the aflatoxins, ochratoxin A (OTA) and zearalenone (ZEN) as well as those that can be rendered fluorescent through derivatization, such as T-2 toxin. The literature describing the applications of cyclodextrins in mycotoxin analysis is reviewed and an attempt to extend the use of cyclodextrins to the detection of labelled T-2 toxin is presented. Twenty cyclodextrins were evaluated for their ability to enhance the fluorescence emission of T-2 toxin derivatized with pyrene-1-carbonyl cyanide (T2-Pyr). This evaluation revealed that heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DIMEB), in particular, enhanced T2-Pyr fluorescence. DIMEB was used as a buffer modifier in a capillary electrophoresis-laser-induced fluorescence (CE-LIF) method for detecting T-2 in maize. Because of the effects that certain cyclodextrins have, especially under aqueous conditions, they may make useful additives for a variety of mycotoxin analytical methods.

A liquid chromatography/tandem mass spectrometry method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, fumonisins (B(1), B(2)), deoxynivalenol, zearalenone, T-2 and HT-2 toxins in maize. A double extraction approach, using a phosphate-buffered solution followed by methanol, was applied to achieve effective co-extraction of the 11 mycotoxins under investigation having quite different polarities and chemical structures. A new multitoxin immunoaffinity column containing antibodies for all these mycotoxins was used to clean up the extract. Detection and quantification of the 11 mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS) using, as chromatographic mobile phase, a linear gradient of methanol/water containing 0.5% acetic acid and 1 mM ammonium acetate. Method performances were quite satisfactory for all tested mycotoxins at contamination levels close to or below the relevant EU maximum permitted or recommended levels. Limits of detection in maize ranged from 0.3 to 4.2 microg/kg. Recoveries higher than 79% were obtained for all tested mycotoxins with relative standard deviations less than 13%.

Scope : The aim of the presented study was to investigate the mycotoxin exposure of Ivorian population related to the consumption patterns of maize, peanuts, millet, and cassava product (attiιkι). Materials and Methods: Maize flour samples ( n = 51) were purchased from all Abidjan local markets, in the south of Ivory Coast, and urine ( n = 99) was collected during the same reference period (July-September 2011) from volunteers living in Abidjan and Daloa cities. Reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS) was used to analyze aflatoxins (AFB 1 , AFB 2 , AFG 1 , and AFG 2 ), ochratoxin A (OTA), fumonisins (FB 1 , FB 2 ), deoxynivalenol (DON), zearalenone (ZEA), and T-2 and HT-2 toxins in maize flour samples, and their relevant biomarkers (AFM 1 , DON, DON + de-epoxydeoxynivalenol (DOM-1), FB 1 , α-zearalenol (ZOL), β-ZOL, and OTA) in urine samples. Results: Critical maize contamination was observed by AFs occurrence (total AFs 4.5 - 330.0 μg/kg) while OTA was found in 13% of samples analyzed. AFM 1 was detected in 40% of urines samples (0.06 - 14.11 ng/ml), OTA in 37% (0.01 - 0.42 ng/ml), FB 1 in 27% (0.07 to 15.31 ng/ml) and, DON was found in 21% of samples at levels up to 10.0 ng/ml. The correlation coefficients (R 2 ) obtained by plotting the percentage of biomarker occurrence (positive samples) versus the frequency of food consumption revealed maize, peanuts, millet and attiιkι were strongly linked to AFB1 and OTA exposure with values of R 2 ranged from 0.462 to 0.956. Conclusion: The present study provided data on mycotoxin risk in Ivory Coast, revealing a frequent co-exposure to the major mycotoxins such as AFs, OTA, and fumonisins, which appeared to be related to the frequency of peanuts, maize, millet and attiιkι consumption. 0

A novel approach is presented herein to study the intestinal absorption of mycotoxins by using a laboratory model that mimics the metabolic processes of the gastrointestinal (GI) tract of healthy pigs. This model was used to evaluate the small-intestinal absorption of zearalenone from contaminated wheat (4.1 mg/kg) and the effectiveness of activated carbon and cholestyramine at four inclusion levels (0.25, 0.5, 1 and 2%) in reducing toxin absorption. Approximately 32% of ZEA intake (247 μg) was released from the food matrix during 6 h of digestion and was rapidly absorbed at intestinal level. A significant reduction of intestinal absorption of ZEA was found after inclusion of activated carbon or cholestyramine, even at the lowest dose of adsorbents, with a more pronounced effect exhibited by activated carbon. In particular, when 2% of activated carbon or cholestyramine was added to the meal the ZEA intestinal absorption was lowered from 32% of ZEA intake to 5 and 16%, respectively. The sequestering effect of both adsorbents took place already during the first 2 h of digestion and persisted during the rest of the experiment. The GI-model is a rapid and physiologically relevant method to test the efficacy of adsorbent materials in binding mycotoxins and can be used to pre-screen mycotoxin/adsorbent combinations as an alternative to animal experiments. © 2003 Elsevier Ltd. All rights reserved.

An improved method for the quantitative determination of aflatoxins (B1, B2, G1, G2), ochratoxin A, fumonisins (B1, B2), zearalenone, deoxynivalenol, nivalenol, T-2 and HT-2 toxins in cereals and derived products, at levels comparable with EU maximum permitted levels, was developed. The effective co-extraction of the mycotoxins under investigation was achieved in 4 min by a double extraction approach, using water followed by methanol. Clean up of the extract was performed by a new multi-toxin immunoaffinity column. Analytical performance characteristics were evaluated through single laboratory validation. Raw wheat and maize, corn flakes and maize snacks were chosen as representative matrices for method validation. The validation assay was carried out at 50, 100 and 150% of EU maximum permitted levels for each mycotoxin. Statistical analysis of the results (ANOVA) provided the within laboratory reproducibility and the error contributions from repeatability, between day effects, and influences from different matrix composition. Recoveries generally higher than 70% were obtained for all tested mycotoxins with relative standard deviation (within laboratory reproducibility) lesser than 37%. Limits of quantification (calculated as the lowest amount of each analyte which could be determined with a precision of 10%) ranged from 1 μg/kg to 30 μg/kg. The trueness of generated data was assessed by analysis of reference materials. The proposed method was proven to be suitable to assess, with a single analysis, compliance of the selected cereal based foods with the EU maximum permitted or recommended levels for all regulated mycotoxins.

Fungal volatile metabolites can be used as an indicator of mycotoxins occurrence in cereals. A rapid, easy-to-perform and non-invasive method using an electronic nose based on metal oxide sensors was developed to distinguish the quality of durum wheat samples in three classes based on the content of the mycotoxin deoxynivalenol (DON): class A ([DON] 2500 μg/kg). Two different approaches were tested by performing 525 analyses of naturally contaminated durum wheat either on whole and ground kernels. Discriminant Function Analysis (DFA) provided calibration models that permitted to classify wheat samples with mean recognition percentages (R) for whole and ground kernels of 69.3% and 86.7%, respectively. Samples belonging to class A were better recognized than those belonging to classes B and C. The e-nose method applied to the analysis of ground wheat showed highest mean recognition percentage (R = 82.1%) in validation. By considering data with recognition percentage rate (Rr) higher than 80% in validation, performances of this method were further improved in terms of either mean recognition percentage (R = 90.1%) and percentage of misclassified samples (4%). An SPME-GC–MS method was also developed that allowed to characterize the pattern of volatile compounds of durum wheat samples. A positive (trichodiene, longifolene, 3-methyl butanal, tridecane, γ-caprolactone, 6,10,14-trimethyl-2-pentadecanone) or negative (hexadecane, 2,3,7-trimethyl-decane, 4,6-dimethyl-dodecane) association with DON content was observed. These results confirm that the developed e-nose method could be used as useful tool for high throughput screening of DON contamination in durum wheat.

Deoxynivalenol (DON) and its main conjugate derivative, DON-3 glucoside (DON-3G), are Fusarium mycotoxins frequently occurring in cereal and cereal-based products destined to human consumption. In this paper, we investigated the stability of DON and DON-3G along the gastric and duodenal-jejunal tract (GI) by submitting a bread food naturally contaminated with both mycotoxins to in vitro digestion experiments. Gastro-duodenal digestion fluids were collected at different time-points along the whole digestion process (gastric and duodenal phases) and mycotoxin content was determined by LC/High Resolution Mass Spectrometry. Our findings show a rather stable behaviour displayed by DON during gastric digestion, whereas a 43% DON decrease was recorded during the passage from the gastric to the duodenal compartment. Ultimately, the apparent and actual bioaccessibility was calculated for DON with values ranging between 34 and 57%, respectively. Data obtained for DON-3G showed no significant differences between the beginning and the end of the gastric phase, whereas a remarked increase of this conjugated mycotoxin was highlighted during the passage in the duodenal compartment. Our results suggest that a conversion of DON in DON-3G is likely to occur during gastro-duodenal digestion of contaminated bread.

[object Object]The gastrointestinal tract is the main target of exposure to mycotoxin fumonisin B1 (FB1), common natural contaminant in food. Previous studies reported that proliferating cells are more sensitive than confluent cells to the toxic effect of FB1. This study aims to investigate, by dose- and time-dependent experiments on human colon proliferating intestinal cell line (HT-29), the modifications induced by FB1 at concentrations ranging from 0.25 to 69 lM. The choice of highest FB1 concentration considered the low toxicity previously reported on intestinal cell lines, whereas the lowest one corresponded to the lower FBs levels permitted by European Commission Regulation. Different functional parameters were tested such as cell proliferation, oxidative status, immunomodulatory effect and changes in membrane microviscosity. In addition FB1- FITC localization in this cell line was assessed by using confocal laser scanning microscopy. Lipid peroxidation induction was the main and early (12 h) effect induced by FB1 at concentrations ranging from 0.5 to 69 lM, followed by inhibition of cell proliferation (up to 8.6 lM), the immunomodulatory effect (up to 17.2 lM), by assessing IL-8 secretion, and increase in membrane microviscosity (up to 34.5 lM). The toxic effects observed in different functional parameters were not dose-dependent and could be the consequence of the FB1 intracytoplasmatic localization as confirmed by confocal microscopy results. The different timescales and concentrations active of different functional parameters could suggest different cellular targets of FB1.

A rapid and accurate method to quantify ochratoxin A (OTA) at ppt (pg/ml) levels in urine has been developed. The method uses commercial immunoaffinity columns for clean-up and reversed phase high-performance liquid chromatography (HPLC) with fluorescence detector for quantification of the toxin. Average recoveries of OTA from human urine spiked at levels from 0.05 ng/ml to 1.0 ng/ml ranged from 88% to 93%, with relative standard deviations (RSDs) between 1% and 8%. Detection limit was 0.005 ng/ml. Out of 41 human urine samples, 25 were found positive to OTA with only one sample exceeding 0.05 ng/ml; the latter originated from a patient affected by karyomegalic interstitial nephritis. The method can be used as a rapid and non-invasive tool to assess human and animal exposure to OTA in epidemiological studies and to establish the possible role of OTA in acute animal intoxications or human end-stage renal diseases.

Grape pomace (pulp and skins) was investigated as a new biosorbent for removing mycotoxins from liquid media. In vitro adsorption experiments showed that the pomace obtained from Primitivo grapes is able to sequester rapidly and simultaneously different mycotoxins. Aflatoxin B-1 (AFB(1)) was the most adsorbed mycotoxin followed by zearalenone (ZEA), ochratoxin A (OTA), and fumonisin B-1 (FB1), whereas the adsorption of deoxynivalenol (DON) was negligible. AFB(1) and ZEA adsorptions were not affected by changing pH values in the pH 3-8 range, whereas OTA and FB1 adsorptions were significantly affected by pH. Equilibrium adsorption isotherms obtained at different temperatures (5-70 degrees C) and pH values (3 and 7) were modeled and evaluated using the Freundlich, Langmuir, Sips, and Hill models. The goodness of the fits and the parameters involved in the adsorption mechanism were calculated by the nonlinear regression analysis method. The best-fitting models to describe AFB(1), ZEA, and OTA adsorption by grape pomace were the Sips, Langmuir, and Freundlich models, respectively. The Langmuir and Sips models were the best models for FB1 adsorption at pH 7 and 3, respectively. The theoretical maximum adsorption capacities (mmol/kg dried pomace) calculated at pH 7 and 3 decreased in the following order: AFB(1) (15.0 and 15.1) > ZEA (8.6 and 8.3) > OTA (6.3-6.9) > FB1 (2.2 and 0.4). Single- and multi-mycotoxin adsorption isotherms showed that toxin adsorption is not affected by the simultaneous presence of different mycotoxins in the liquid medium. The profiles of adsorption isotherms obtained at different temperatures and pH and the thermodynamic parameters (Delta G degrees, Delta H degrees, Delta S degrees) suggest that mycotoxin adsorption is an exothermic and spontaneous process, which involves physisorption weak associations. Hydrophobic interactions may be associated with AFB(1) and ZEA adsorption, whereas polar noncovalent interactions may be associated with OTA and FB1 adsorption. In conclusion, this study suggests that biosorption of mycotoxins onto grape pomace may be a reasonably low-cost decontamination method.

A single isolates ofFusarium graminearum Schwabe KF 366 andFusarium culmorum (W.G.Sm.) Sacc. KF 365 were used to infect 10 genotypes (9 lines and one cultivar) of winter triticale, 1 rye cultivar and 1 wheat cultivar, and amounts of mycotoxins in kernels were analysed at the same stage of development. One genotype of triticale CHD 353/79 and rye “Chodan” were found to be most resistant towards both species infection with lowest amount of mycotoxins (deoxynivalenol) content in kernels and also the lowest yield reduction. The most susceptible line CZR 142 cumulated in kernels about ten times higher amount of mycotoxins (up 53 mg DON/kg and 16 mg 3AcDON/kg, and 5 mg zearalenone/kg). GenerallyF, culmorum formed higher level of mycotoxins in kernels of infected heads thanF. graminearum. In kernels of more susceptible genotypes except deoxynivalenol, 3 acetyldeoxynivalenol and zearalenone also were present.

Microdochium nivale (Fusarium nivale) was found to be frequently occuring in Poland pathogen of small grain cereals heads, causing symptoms similar to those observed after infection ofFusarium species. In consecutive years since 1985 till 1989 the following percentage of wheat and rye ears infected withM. Nivale and withFusarium head blight symptoms was found: 34%, 21%, 42%, 9%, 46% (wheat) and 57%, 43%, 65%, 4%, 47% (rye) heads.However, in naturally infected rye and wheat samples (kernels and chaff), we did not detect toxins usually present in samples infected with fungi of genusFusarium - such as deoxynivalenol and derivatives. TypicalFusarium trichothecene metabolites were also not present in cultures of 11M. nivale strains, growing 3-5 weeks on rice (45% water content) at 20°C. Cultures of two typical isolates on wheat grain (strain KF 1124) and on rice (KF 245) were found to be non toxic to broiler chickens when present in amount 20-40% in their diet. It can be concluded thatM. nivale (F. nivale) representatives in Poland did not produce toxic metabolites neither under laboratory condition nor after cereal ears infection under field conditions.

Fusarium tricinctum strain (KF 260) isolated from wheat in Poland, produced on maize cultures a compound (C11H14O5, MW226) toxic toArtemia salina. The compound, identified by various spectroscopical tecniques as 5-hydroxy-4-methoxy-6,8a-dimethyl-6, 7-dihydro-2H, 8aH-pyrano/2, 3-b/pyran-2-one, was identical to chlamydosporol, a mycotoxin recentlydiscovered in cultures ofF._ chlamydosporum isolated from rice. The compound showed inhibitory effect on human lymphocyte proliferation at concentration of 25 μg/mL. LD50 onA. salina was 56 μg/mL.

The benefits of using rapid qualitative methods to verify compliance of food and feed with legislation requirements include user-friendly format, the possibility of detection without expensive instrumentation, rapid response and affordable price. Prior to their use, however, the methods have to pass validation experiments, in order to assess their performance profile. An experimental protocol for in-house validation of a screening immunoassay has been designed and applied to evaluate performance characteristics of a multiplex dipstick kit for the determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat and maize. The test is intended for screening of cereals on the presence/absence of these mycotoxins at maximum permitted levels established by European legislation or target levels. The response of the measurement is determined with a reader device. Samples classified as negative are considered as compliant, whereas positive samples need to be re-analysed with confirmatory methods. The in-house validation design consisted of three steps, namely (1) estimating the precision of the method including "between day" effects and influences from different varieties of the matrices, (2) establishing robust cutoff values for the dipstick response at target mycotoxin levels assuming an acceptable rate of false negative results of 5 % and (3) assessment of the rate of false positive results of blank samples and samples containing the target analytes below the legal limits. The total precision expressed as relative standard deviation and determined individually for each analyte/concentration/matrix combination varied from 9 to 30 % and was considered as acceptable. In 17 out of 28 cases, the repeatability standard deviation was the most important factor. The predominance of the repeatability compared to the other factors (matrix, days) was an indicator for the ruggedness of the assay. The validation study demonstrated that the test was able to differentiate blank samples from samples contaminated at target mycotoxin levels with a false positive rate lower than 6 %. Considering realistic mycotoxin occurrence in European samples, significant economical benefits can be expected when using the test under real-world conditions.

Liquid chromatography coupled with single or tandem mass spectrometry (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 the European Committee for Standardization and the Association of Analytical Communities. A proficiency test (PT) was conducted for the simultaneous determination of up to 11 mycotoxins (aflatoxin B 1 (AFB 1 ), aflatoxin B 2 (AFB 2 ), aflatoxin G 1 (AFG 1 ), aflatoxin G 2 (AFG 2 ), ochratoxin A (OTA), deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEA), fumonisin B 1 (FB 1 ) and fumonisin B 2 (FB 2 )) 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. Each participant received the following: instructions; a comprehensive questionnaire; a mixed mycotoxins calibration solution; a spiking solution (AFB 1 , AFB 2 , AFG 1 and AFG 2 , OTA, DON, T-2, HT-2, ZEA, FB 1 and FB 2 ); and 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. 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%, FB 1 50% and 30%, FB 2 52% and 38%, ZEA 68% and 64%, T-2+HT-2 toxins 82% and 85%, OTA 58% and 60%, AFB 1 56% and 62%, AFG 1 73% and 84%, AFB 2 40% and 78%, AFG 2 64% and 78%, respectively. The poorest performance (|z| >3) was obtained for FB 1 (31%), FB 2 (32%), AFB 1 (32%) and AFB 2 (32%) in contaminated maize and for DON (35%), FB 1 (63%) and FB 2 (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 FB 1 and 163% for FB 2 ). 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 inter-laboratory validation. The results of this PT and the relevant method’s details can be useful to identify methodology strengths and weaknesses.

A sensitive and accurate fluorescence polarization (FP) immunoassay has been developed for the determination of ochratoxin A (OTA) in naturally contaminated wheat samples. A fluorescein-labeled OTA tracer was synthesized, and its binding response with three monoclonal antibodies was tested. The most sensitive competitive FP immunoassay showed an IC50 value of 0.48 ng/mL with a negligible cross-reactivity for ochratoxin B (1.7 %) and no cross-reactivity with other mycotoxins commonly occurring in wheat. The wheat sample was extracted with acetonitrile/water (60:40, v/v) and purified by a rapid solid-phase extraction procedure using an aminopropyl column prior to the FP immunoassay. The overall time of analysis was less than 20 min. The average recovery from spiked wheat samples (3 to 10 mu g/kg) was 87 %, with relative standard deviations generally lower than 6 %. Limits of detection and quantification were 0.8 and 2.0 mu g/kg, respectively. The trueness of the method was assessed by using two reference materials for OTA showing good accuracy and precision. A good correlation (r = 0.995) was observed between OTA contamination of 19 naturally contaminated wheat samples analyzed by both FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up used as reference method. These results show that the developed FP method is suitable for high-throughput screening, as well as for reliable quantitative determination of OTA in wheat at level far below the EU regulatory limits.

Fifty commercial corn hybrids with different endosperm characteristics, vegetative cycle length and cross class grown in the same geographical area (Cordoba Province, Argentina) were analysed for fumonisin accumulation. All hybrids analysed showed fumonisin B(1) and B(2) contamination ranging from 185 to 27,050 ng/g for FB(1) and from 40 to 9950 ng/g for FB(2). Although most of the hybrids analysed had flint-type endosperm, two hybrids with dent-type endosperm (e.g. Prozea 10 and AX 746) showed the highest level of fumonisin (37,000 ng/g) and more FB(2) than FB(1) (FB(2)/FB(1) ratio 2.42), respectively. There was no correlation between fumonisin concentration and length of the vegetative cycle. Among 18 hybrids examined for Fusarium species contamination there was also no correlation between fumonisin contamination and the level of infection with Fusarium species (Section Liseola). Eighteen hybrids showed fumonisin levels lower than 1000 ng/g. This result suggests that there is some possibility of selecting hybrids resistant or less susceptible to fumonisin and Fusarium contamination.

The distribution of Fusarium species (Liseola section) and fumonisins B 1 , B 2 , and B 3 (FB 1 , FB 2 , and FB 3 ) in corn at the field stage to harvest time has been investigated. Corn samples were obtained from the 45th day after flowering to harvest stage, with 15 day intervals. A good correlation between fungal species and fumonisin contamination was observed. In particular, fumonisin contamination was higher in samples with fungal infection represented mainly by Fusarium moniliforme and Fusarium proliferatum, two well-known producers of fumonisins. Low levels of fumonisins were detected at the first and second samplings, when mainly Fusarium subglutinans, a fumonisin nonproducer species, was recorded. The three samplings after physiological maturity showed predominant F. moniliforme and F. proliferatum infection and a considerable contamination with fumonisins. In these samples the levels of FB 1 averaged above 1 μg/g and FB 2 and FB 3 levels showed similar values but lower than FB 1 values. This is the first report of fumonisins in Argentinian corn at field stages.

Fumonisins are mycotoxins mainly produced by Fusarium moniliforme, one of the most prevalent seed-borne fungi of maize. Strains of Fusarium moniliforme isolated from cereals in Europe produced in cultures high levels (up to 4 mg/g) of fumonisins B1 and B2 (FB1 and FB2). FB1 and FB2 have been found in maize and maize-based foods and feeds in most European countries. In Italy these mycotoxins have been directly involved in a fatal outbreak of equine leukoencephalomalacia, and have been found at different levels in most maize-based food products, including polenta--a staple food in a region with high incidence of esophageal cancer. The European Commission, Community Bureau of Reference (BCR) and Measurements and Testing Programme (M&T), has undertaken two consecutive intercomparison studies, involving over 20 laboratories from 12 European countries, to improve the quality of fumonisins analysis at European level. The first study consisted in the evaluation of the determination of fumonisins in an unknown solution, while the second one involved the analytical methodology for fumonisins in contaminated and blank maize samples. A definite improvement in the performance of the different laboratories has been observed in the second study. In view of the production of reference materials certified for their fumonisins contents, new analytical procedures need to be developed.

Deoxynivalenol, T-2 and HT-2 toxins are mycotoxins frequently occurring in cereals and cereal-based products along with their conjugated forms. In this paper, we provide insights into the fate of deoxynivalenol, T-2 and HT-2 toxins and their glucoside derivatives during bread making, using naturally contaminated wheat flour. High-resolution mass spectrometry was used to assess the extent of degradation of the three mycotoxins during bread baking and to identify some glucoside conjugates, namely deoxynivalenol, T-2 and HT-2 mono-glucosides, detected both in the flour and in the respective breads. Our findings show deoxynivalenol's levels markedly increased upon baking, whereas those of HT-2 and T-2 toxins were decreased in the final bread with special regard to the T-2 toxin.

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 μg kg(-1), respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 μg 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.

European intake estimates indicate that the presence of HT-2 and T-2 toxins in cereals, mainly in oats, can be of concern for human health. Therefore, the development of sensitive, rapid and reliable methods for determining these mycotoxins in cereals, in particular oats, has high priority. A rapid ultra-performance liquid chromatographic (UPLC) method has been developed for the simultaneous determination of HT-2 and T-2 toxins in oats and wheat at μg kg(-1) level. Ground samples were extracted with methanol/water (90:10, v/v) and the diluted extracts were cleaned up through immunoaffinity columns. HT-2 and T-2 toxins were separated and quantified by UPLC with photodiode array (PDA) detector (λ=202 nm) in less than 5 min. Mean recoveries from blank oats samples spiked with HT-2 and T-2 toxins at levels of 50-1000 μg kg(-1) ranged from 87 to 96%, with relative standard deviations (RSDs) lower than 7%; mean recoveries from wheat spiked with HT-2 and T-2 toxins at levels of 25-100 μg kg(-1) ranged from 91 to 103%, with RSDs lower than 5%. The limit of detection of the method was 8 μg kg(-1) for both toxins (signal-to-noise ratio 3:1). The method was successfully applied to the analysis of HT-2 and T-2 toxins in naturally contaminated oats and wheat samples. A good correlation was found by comparative analysis of naturally contaminated samples of oats (r=0.9985) and wheat (r=0.9058) using the proposed method or a reliable HPLC method with fluorescence detection after pre-column derivatization with 1-anthroylnitrile.

Acuminatopyrone, a recently described metabolite of Fusarium acuminatum was shown to be produced by several strains of Fusarium chlamydosporum and F. tricinctus isolated from various substrates in different countries. The structure of acuminatopyrone has been revised on the basis of NOEDIF and HETCOR nmr experiments, and shown to be 4-methoxy-7,8-dimethyl-2H-pyrano[4,3-b]pyridin-2-one. This structure is biosynthetically consistent with that of chlamydosporol and related compounds that are produced by the same fungal species

A sensitive and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods. Samples were extracted with a mixture of acetonitrile/water (84:16, v/v) and cleaned up through a polymeric solid-phase extraction column. Detection and quantification of the nine mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS), using fully (13)C-isotope-labelled mycotoxins as internal standards. The method was validated in-house for five different cereal processed products, namely barley, oat and durum wheat flours, rye- and wheat-based crisp bread. Recoveries and repeatability of the whole analytical procedure were evaluated at contamination levels encompassing the EU maximum permitted levels for each tested mycotoxin. Recoveries ranged from 89 to 108% for deoxynivalenol, from 73 to 114% for aflatoxins, from 85 to 114% for T-2 and HT-2 toxins, from 64 to 97% for zearalenone, from 74 to 102% for ochratoxin A. Relative standard deviations were less than 16% for all tested mycotoxins and matrices. Limits of detection (signal-to-noise ratio 3:1) ranged from 0.1 to 59.2 µg/kg. The trueness of the results obtained by the proposed method was demonstrated by analysis of reference materials for aflatoxins, deoxynivalenol, zearalenone. The use of inexpensive clean-up cartridges and the increasing availability of less expensive LC/MS/MS instrumentation strengthen the potential of the proposed method for its effective application for reliable routine analysis to assess compliance of tested cereal products with current regulation.