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Driven by increasing temperatures and the higher incidences of heat waves during summer, an increased incidence of Aspergillus flavus next to Fusarium verticillioides in European maize can be expected. In the current study, we investigated the interaction between both species. Colonies of A. flavus/F. verticillioides were grown in a single culture, in a dual culture, and in a mixed culture. The growth rate of A. flavus and F. verticillioides grown in a dual or mixed culture with the other species was clearly slower compared to the growth rate in a single culture. Mycotoxin production was in most cases negatively affected by dual or mixed inoculation. In planta, a dual inoculation resulted in reduced lesions of A. flavus, whereas the lesion size and toxin production of F. verticillioides were unaffected in the presence of A. flavus. The lesions as a result of a mixed inoculation were 112% bigger than a single A. flavus inoculation and 9% smaller than a single F. verticillioides inoculation. The fumonisin levels were 17% higher compared to a single inoculation. In case A. flavus was present two days before F. verticillioides, the lesion size of F. verticillioides was 55% smaller compared to a single F. verticillioides inoculation, and fumonisin production was almost completely inhibited. The interaction between A. flavus and F. verticillioides is highly dynamic and depends on the experimental conditions, on the variables measured and on the way they colonize the host, in two inoculation points, simultaneously in one inoculation point, or sequentially one species colonizing an existing lesion made by the other., (© 2021. Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature.)

Maize silage, which in Europe is the main feed for dairy cattle in winter, can be contaminated by mycotoxins. Mycotoxigenic Fusarium spp. originating from field infections may survive in badly sealed silages or re-infect at the cutting edge during feed-out. In this way, mycotoxins produced in the field may persist during the silage process. In addition, typical silage fungi such as Penicillium spp. and Aspergillus spp. survive in silage conditions and produce mycotoxins. In this research, 56 maize silages in Flanders were sampled over the course of three years (2016-2018). The concentration of 22 different mycotoxins was investigated using a multi-mycotoxin liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and the presence of DNA of three Fusarium spp. ( F. graminearum , F. culmorum and F. verticillioides ) was analyzed in a selection of these samples using quantitative polymerase chain reaction (qPCR). Every maize silage contained at least two different mycotoxins. Nivalenol (NIV) and deoxynivalenol (DON) were the most prevalent (both in 97.7% of maize silages), followed by ENN B (88.7%). Concentrations often exceeded the EU recommendations for DON and zearalenone (ZEN), especially in 2017 (21.3% and 27.7% of the maize silages, respectively). No correlations were found between fungal DNA and mycotoxin concentrations. Furthermore, by ensiling maize with a known mycotoxin load in a net bag, the mycotoxin contamination could be monitored from seed to feed. Analysis of these net bag samples revealed that the average concentration of all detected mycotoxins decreased after fermentation. We hypothesize that mycotoxins are eluted, degraded, or adsorbed during fermentation, but certain badly preserved silages are prone to additional mycotoxin production during the stable phase due to oxygen ingression, leading to extremely high toxin levels.

Forage maize is often infected by mycotoxin-producing Fusarium fungi during plant growth, which represent a serious health risk to exposed animals. Deoxynivalenol (DON) and zearalenone (ZEN) are among the most important Fusarium mycotoxins, but little is known about the occurrence of their modified forms in forage maize. To assess the mycotoxin contamination in Northern Germany, 120 natural contaminated forage maize samples of four cultivars from several locations were analysed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) for DON and ZEN and their modified forms deoxynivalenol-3-glucoside (DON3G), the sum of 3- and 15-acetyl-deoxynivalenol (3+15-AcDON), α- and β-zearalenol (α-ZEL, β-ZEL). DON and ZEN occurred with high incidences (100 and 96%) and a wide range of concentrations, reaching levels up to 10,972 and 3910 µg/kg, respectively. Almost half of the samples (46%) exceeded the guidance value in complementary and complete feeding stuffs for ZEN (500 µg/kg), and 9% for DON (5000 µg/kg). The DON related mycotoxins DON3G and 3+15-AcDON were also present in almost all samples (100 and 97%) with amounts of up to 3038 and 2237 µg/kg and a wide range of concentrations. For the ZEN metabolites α- and β-ZEL lower incidences were detected (59 and 32%) with concentrations of up to 423 and 203 µg/kg, respectively. Forage maize samples were contaminated with at least three co-occurring mycotoxins, whereby 95% of all samples contained four or more mycotoxins with DON, DON3G, 3+15-AcDON, and ZEN co-occurring in 93%, together with α-ZEL in 57% of all samples. Positive correlations were established between concentrations of the co-occurring mycotoxins, especially between DON and its modified forms. Averaged over all samples, ratios of DON3G/DON and 3+15-AcDON/DON were similar, 20.2 and 20.5 mol%; cultivar-specific mean ratios ranged from 14.6 to 24.3 mol% and 15.8 to 24.0 mol%, respectively. In total, 40.7 mol% of the measured DON concentration was present in the modified forms DON3G and 3+15-AcDON. The α-ZEL/ZEN ratio was 6.2 mol%, ranging from 5.2 to 8.6 mol% between cultivars. These results demonstrate that modified mycotoxins contribute substantially to the overall mycotoxin contamination in forage maize. To avoid a considerable underestimation, it is necessary to analyse modified mycotoxins in future mycotoxin monitoring programs together with their parent forms.

Together with conducive climatic factors, poor pre-harvest practices of ethnic small-holder farmers are a major cause of the contamination of maize by Fusarium verticillioides and fumonisins. The proliferation of this field pathogen and the accumulation of its mycotoxins in post-harvest maize caused by ethnic post-harvest practices of subsistence farms have received little attention. Therefore, this study aimed to evaluate the impact of traditional harvest and post-harvest practices on the proliferation of F. verticillioides and fumonisins contamination in post-harvest maize of two ethnic groups: Ede and Kinh from the central highlands of Vietnam. In parallel with analysis, a survey on harvest and post-harvest practices of these farmers was conducted from late December 2017 to early January 2019. As a result, four effective post-harvest practices at mitigating the contamination were defined: (1) removal of damaged cobs at harvest, (2) transport of maize home after harvest, (3) shelling maize away from fields, and (4) drying maize on cement yards. These practices were better implemented by Kinh households than Ede households reducing the post-harvest contamination of maize with F. verticillioides and fumonisin B 1 (FB 1 ), FB 2 , and FB 3 . Nevertheless, there is still room for improvement with respect to inadequate open-air drying method, poor storage infrastructure, and poor moisture content management as these correlated to the proliferation of F. verticillioides. Finally, the presence of fumonisins together with aflatoxins in some samples at the storage phase might cause a severe impact on human health., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Although previous studies have reported the use of nixtamalization for mycotoxins reduction in maize, the efficacy of calcium hydroxide and other nixtamalization cooking ingredients for mycotoxin reduction/decontamination in sorghum and other cereals still need to be determined. The current study investigated the effect of five nixtamalization cooking ingredients (wood ashes, calcium hydroxide, sodium hydroxide, potassium hydroxide, and calcium chloride) on the reduction of Fusarium mycotoxins in artificially contaminated maize and sorghum using liquid chromatography-tandem mass spectrometry. All tested cooking ingredients effectively reduced levels of mycotoxins in the contaminated samples with reduction initiated immediately after the washing step. Except for the calcium chloride nixtamal , levels of fumonisin B 1 , B 2, and B 3 in the processed sorghum nixtamal samples were below the limit of detection. Meanwhile, the lowest pH values were obtained from the maize (4.84; 4.99), as well as sorghum (4.83; 4.81) nejayote and nixtamal samples obtained via calcium chloride treatment. Overall, the results revealed that the tested cooking ingredients were effective in reducing the target mycotoxins. In addition, it pointed out the potential of calcium chloride, though with reduced effectiveness, as a possible greener alternative cooking ingredient (ecological nixtamalization) when there are environmental concerns caused by alkaline nejayote .

Fusarium mycotoxins and their derivatives are frequently detected in freshly harvested forage maize. This study assessed the time course effects during ensiling of forage maize on the fate of Fusarium mycotoxins, using laboratory-scale silos and artificially contaminated raw material. A multi-mycotoxin liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was used to determine the levels of deoxynivalenol (DON), zearalenone (ZEN) and their derivatives DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, deepoxy-DON, α-zearalenol and β-zearalenol. A significant increase of DON was observed during ensiling, whereas the levels of DON-3-glucoside and its acetylated forms proportionally decreased. In contrast, levels of ZEN, α-zearalenol and β-zearalenol were not affected by the ensiling process. Based on these findings, ensiling is not a practical method for reducing the total amount of Fusarium mycotoxins present at harvest.

Effective management of mycotoxins rely on stringent regulation and routine surveillance of food/feed commodities via efficient analysis, hence the continuous need for improved methods. The present study developed, optimized and validated a modified pressurized hot water extraction (PHWE) method for the simultaneous extraction of multi-mycotoxins from maize and subsequent quantification on LC-MS/MS. The PHWE system was modified using ethanol (EtOH) as a cosolvent, while a numerical modelling approach, the central composite design (CCD), was adopted for the optimization of the extraction conditions. Using the optimized method, it was possible to effectively extract and quantify 15 different mycotoxins from maize in a single step with satisfactory linearities (0.986-0.999), recoveries (14-124%) and other associated method validation parameters. Further efficacious application of the method to real samples re-affirmed the prospects of PHWE as a suitable, cost-effective and greener alternative to traditional methods of mycotoxin extraction., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mycotoxins are fungal secondary metabolites frequently affecting agronomical crops and consequently imposing a major challenge for food safety and public health. In this study, a total of 67 raw cereals (55 maize and 12 sorghum) were collected from the market of Togo. The samples were investigated on the occurrence of 21 mycotoxins using state-of-the-art high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The most frequent occurring mycotoxins were fumonisins (88 and 67% for maize and sorghum respectively) with concentrations ranging from 101 to 1838 μg/kg for maize and 81.5 to 361 μg/kg for sorghum, respectively. Aflatoxin B1 was detected in 38% of the maize samples with maximum contamination levels of 256 μg/kg, and 25% of the sorghum samples (range 6-16 μg/kg). The concentrations of aflatoxins were high in maize, with some cases exceeding the maximum legislative limits (EU) for unprocessed maize placed on the market. In addition to these high contamination levels, the co-occurrence of three classes of mycotoxins (i.e., aflatoxins, fumonisins, and trichothecenes) was observed in this study. For the first time, the multi-mycotoxins occurrence in agronomical crops in Togo was reported.

The selective and sensitive analysis of mycotoxins in highly complex feed matrices is a great challenge. In this study, the suitability of Orbitrap TM -based high-resolution mass spectrometry (HRMS) for routine mycotoxin analysis in complex feeds was demonstrated by the successful validation of a full MS/data-dependent MS/MS acquisition method for the quantitative determination of eight Fusarium mycotoxins in forage maize and maize silage according to the Commission Decision 2002/657/EC. The required resolving power for accurate mass assignments (<5 ppm) was determined as 35,000 full width at half maximum (FWHM) and 70,000 FWHM for forage maize and maize silage, respectively. The recovery (R A ), intra-day precision (RSD r ), and inter-day precision (RSD R ) of measurements were in the range of 94 to 108%, 2 to 16%, and 2 to 12%, whereas the decision limit (CCα) and the detection capability (CCβ) varied from 11 to 88 µg/kg and 20 to 141 µg/kg, respectively. A set of naturally contaminated forage maize and maize silage samples collected in northern Germany in 2017 was analyzed to confirm the applicability of the HRMS method to real samples. At least four Fusarium mycotoxins were quantified in each sample, highlighting the frequent co-occurrence of mycotoxins in feed.

Fusarium graminearum can cause Giberella Ear Rot (GER) and seedling blight in maize, resulting in major yield losses. Besides GER, the infected grains are consequently contaminated with multiple mycotoxins of F. graminearum . Zearalenone and trichothecenes, such as deoxynivalenol and its acetylated forms, are among the major mycotoxins associated with F. graminearum infection in maize. In the current work, we explored the effect of the endophytic fungal genera of Epicoccum and Sordaria , to control F. graminearum infection in comparative trials with Piriformospora spp., an elusive endophytic genus. Furthermore, we investigated the effect of these endophytes on zearalenone, deoxynivalenol, and 15-acetyldeoxynivalenol levels using in vitro and in planta assays. As plants are endowed with several detoxification mechanisms comprising e.g., glucosylation of trichothecenes, the effect of the isolated fungal endophytes on the deoxynivalenol-3-glucoside level was also assessed. In general, results showed a considerable variability in the antifungal activity, both among species and among isolates within one species. Additionally, the effect on mycotoxin levels was variable, and not necessarily related to the antifungal activity except for zearalenone levels which were consistently reduced by the endophytes. These results highlight the great potential of certain endophytic fungal strains as new biocontrol agents in agricultural science.

Aims: The present study was conducted to evaluate the antagonistic effect of Bacillus velezensisNRRL B-23189 towards Penicillium roqueforti s.s. and Penicillium paneum (designated together as P. roqueforti s.l.) in silage conditions., Methods and Results: Corn silage conditions were simulated in vitro, and the impact of B. velezensis culture supernatant or cell suspension on P. roqueforti s.l. growth and roquefortine C production was evaluated. The antagonism was promising, but growth of B. velezensis in corn silage infusion was poor. Additionally, an in vivo experiment was carried out with mini-silos containing a mixture of perennial ryegrass and white clover inoculated with P. roqueforti s.l. The applied B. velezensis cell suspension was unsuccessful in reducing P. roqueforti s.l. numbers, but did not compromise the silage acidification., Conclusions: Although the antagonism observed in vitro was promising, the applied B. velezensis cell suspension could not live up to the expectations in vivo., Significance and Impact of the Study: To our knowledge, the present study is the first one evaluating the antagonistic properties of B. velezensis towards toxigenic moulds in silage conditions, offering a good base for further research., (© 2018 The Society for Applied Microbiology.)

A fundamental step in addressing the global problem of mycotoxins is the development of highly sensitive, multi-class extraction and detection methods. This constitutes a field of research that has in recent years enjoyed a steady advance. Such methods, generally based on liquid chromatography coupled to mass spectrometry, are widely reported successfully detecting various mycotoxins in different food and feed samples. In this work, an innovative approach to multi-class mycotoxin control is proposed, offering specific advantages: a broader inclusion of more mycotoxin classes, robust and thorough extraction for all target compounds despite their varied chemical properties, and determination of all analytes from a single injection. The method involved the extraction and quantification of the main mycotoxins produced by Aspergillus, Fusarium, and Penicillium fungi, as well as their reported derivatives, together with 12 other compounds most commonly produced by Claviceps purpurea. The popularly reported QuEChERS technique has been reduced to a simple "salting-out liquid-liquid extraction" (SO-LLE) to obtain the most efficient extraction of the aforementioned mycotoxin classes in a very short time. This is in particular extremely important in ensuring correct determination of individual ergot alkaloids, for which short and robust sample preparation as well as short analytical sequences were key for minimizing the epimerization during analysis. The analyses of wheat and maize samples were performed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry. Matrix-matched calibration curves were established and limits of quantification were below the maximum levels established by the EU regulation. The precision (repeatability and intermediate precision) was lower than 13% in all cases and recoveries ranged between 60 and 98% in maize and between 62 and 103% in wheat, fulfilling the current legislation. The method was applied to study the co-occurrence of these mycotoxins in wheat (n = 13) and maize (n = 15) samples from six European countries. A successful quantification of 23 different mycotoxins, from all major classes, in 85% of wheat and 93% of maize samples was achieved.

Aflatoxins are the most potent genotoxic and carcinogenic mycotoxins. To date, research has only focused on the presence of free aflatoxins in agricultural commodities. Therefore, the main objective of this study was to investigate the occurrence of possible modified aflatoxins in maize. Different hydrolysis methods were applied to convert modified mycotoxins into their free aflatoxins. Eighteen aflatoxin-contaminated maize samples were incubated with potassium hydroxide, trifluoromethanesulfonic acid and several enzymes to induce hydrolysis. Potassium hydroxide caused a total reduction of aflatoxins, while trifluoromethanesulfonic acid did not lead to an increase in free aflatoxins, neither did treatment with a protease. However, α-amylase and cellulase incubation caused significant increases in the total free aflatoxin content, 15 ± 8% and 13 ± 5%, respectively. These results show that a small proportion of aflatoxins could be associated to matrix substances in plants. Consequently, hydrolysis could occur during food processing and during mammalian digestion, leading to an underestimation of the total aflatoxin content., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Beer, a beverage consumed throughout the world, is mainly derived from cereals. In this study, fungal and mycotoxin contamination, as well as the physicochemical properties of maize-based opaque beer (umqombothi) obtained from the Gauteng province of South Africa, was investigated. The mean water activity, pH and total titratable acidity of the analysed beer samples were 0.91, 3.76 and 1.20% lactic acid, respectively. The investigation revealed Aspergillus, Penicillium, Phoma and Saccharomyces as the predominant fungal genera with a mean fungal load of 3.66 × 10 5  CFU/mL. Among the mycotoxigenic fungal species recovered, Aspergillus flavus had the highest incidence of 26%. Previously unreported strains such as P. chrysogenum strain AD25, A. sydowii strain AD 22 and A. tritici strain AD 11 were found. Furthermore, mycotoxin quantitative analysis via liquid chromatography-tandem mass spectrophotometry showed that deoxynivalenol was the dominant mycotoxin occurring in 84% of the samples. This was followed by enniatin B that occurred in 75% of samples ranging from 12 to 44 μg/L and fumonisin B 1 (FB 1 ) (incidence of 53% at a maximum level of 182 μg/L). Generally, there was low occurrence aflatoxins, whereas T-2, HT-2, nivalenol, zearalenone, 3- and 15-acetyl-deoxynivalenol were not detected. All the samples analysed had safe levels of mycotoxins tested but were contaminated by at least two mycotoxins that could pose some additive or synergistic health effects among consumers. On average: a 60 kg adult consuming 1-6 L/day of the beer was exposed to FB 1  + FB 2 at an estimated 2.20-13.20 μg/kg body weight/day. These values were far above the maximum tolerable daily intake of 2 μg/kg bw/day established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The study demonstrates that consumption of umqombothi can significantly enhance dietary exposure to multiple mycotoxins among consumers, and therefore accentuates the need for strategies aimed at reducing toxigenic fungal colonization and mycotoxin contamination in the beer processing chain., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A sensitive tool for simultaneous quantitative determination of three analytes in one single well of a microtiter plate is shown for the first time. The developed technique is based on use of colloidal quantum dot enrobed into a silica shell (QD@SiO2) derivatives as a highly responsive label. Silica-coated quantum dots were prepared and subsequently modified via the co-hydrolysis with tetraethylorthosilicate (TEOS) and various organosilane reagents. Different surface modification schemes were compared in terms of applicability of the obtained particles for the multiplex immunoassay, e.g. stability and simplicity of their conjugation with biomolecules. As model system a multiplex immunosorbent assay for screening of three mycotoxins (deoxynivalenol, zearalenone and aflatoxin B1) in cereal-based products was realized via a co-immobilization of three different specific antibodies (anti- deoxynivalenol, anti-zearalenone and anti-aflatoxin B1) in one single well of a microtiter plate. Mycotoxins were simultaneously determined by labelling their conjugates with QD@SiO2 emitting in different parts of the visible spectrum. The limits of detection for the simultaneous determination were 6.1 and 5.3, 5.4 and 4.1, and 2.6 and 1.9µgkg(-1) for deoxynivalenol, zearalenone and aflatoxin B1 in maize and wheat, respectively. As confirmatory method, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We report an unusual aflatoxin profile in maize and groundnuts from Malawi, with aflatoxin G1 found routinely at equal or even higher levels than aflatoxin B1. Aflatoxin B1 (AFB1) ratio in a contaminated sample is generally greater than 50% of total aflatoxin (sum of aflatoxin B1, B2, G1, and G2). In Malawi, the aflatoxin occurrence ratios were determined by examining LC-MS/MS and HPLC fluorescence detection (FLD) data of 156 naturally contaminated raw maize and 80 groundnut samples collected in 2011 and 2012. Results showed that natural aflatoxin occurrence ratio differed. In 47% of the samples, the concentration of AFG1 was higher than that of AFB1. The mean concentration percentages of AFB1/AFB2/AFG1/AFG2 in reference to total aflatoxins were found to be 47:5:43:5%, respectively. The AFG1 and AFB1 50/50 trend was observed in maize and groundnuts and was consistent for samples collected in both years. If the AFB1 measurement was used to check compliance of total aflatoxin regulatory limit set at 10, 20, 100, and 200 μg/kg with an assumption that AFB1≥50% of the total aflatoxin content, 8, 13, 24, and 26% false negative rates would have occurred respectively. It is therefore important for legislation to consider total aflatoxins rather than AFB1 alone.

Maize is one of the major staple foods of Sub-Saharan Africa and is consumed as whole or dehulled grain. In this region, where the environmental conditions favour fungal growth and mycotoxin production, the majority of the population are subsistence consumers who, unfortunately, have little or no access to mycotoxin testing of their food. In an attempt to develop feasible reduction strategies in dietary mycotoxin exposure of the population, a three-factorial design experiment was conducted to examine and compare the efficacy of hand sorting, flotation, dehulling and combinations thereof in removing naturally occurring aflatoxins, fumonisins, nivalenol, deoxynivalenol and alternariol in shelled white maize. Regression analysis was used to determine the significant (p < 0.05) process variables on the removal of mycotoxins from the maize. Results from this experiment indicated that hand sorting had the greatest effect on mycotoxin removal, while flotation yielded the least effect. In particular hand sorting left < 6% of aflatoxin B1 and < 5% of fumonisin B1. Based on these results, hand sorting of maize grains is being recommended as a last line of defence against mycotoxin exposure among subsistence consumers.

Humans are naturally and frequently exposed to a multitude of mycotoxins, but health risk assessments are usually performed on individual mycotoxins, which may underestimate the total risks. In this study, we assessed for the first time the cumulative health risks of concomitant exposure via dietary intake (DI) to multiple mycotoxins, namely deoxynivalenol (DON) and its acetyl derivatives of 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON), based on the concentration addition (CA) concept. A cross-sectional study was conducted in seven districts in Shanghai, China with 1269 participants and 330 wheat and maize samples analyzed. After probabilistic analysis using Monte Carlo simulation, the results showed no health risks to the population in Shanghai considering individual mycotoxins. However, if the cumulative health risks were calculated based on the combined consideration of DON with either 3-ADON or 15-ADON or both, the DI values in 95th percentile were up to 1087 ng/kg body weight/day, exceeding the Provisional Maximum Tolerable Daily Intake (PMTDI) of 1000 ng/kg body weight/day and hence representing potential health risks to the population in Shanghai. The integrated study proposed here could be a model strategy for cumulative health risk assessment on the co-occurring hazards in the fields of food safety combined with environmental contaminants.

A fluorescence-based immunochromatographic assay (ICA) for fumonisin B1 (FB1) that employs conjugates of fluorescent microspheres and monoclonal antibodies (FM-mAbs) as detection reporters is described. The ICA is based on the competitive reaction between FB1-bovine serum albumin (BSA; test line) and the target FB1 for binding to the FM-mAb conjugates. A limit of detection (LOD) for FB1 of 0.12 ng/mL was obtained, with an analytical working range of 0.25-2.0 ng/mL (corresponding to 250-2000 μg/kg in maize flour samples, according to the extraction procedure). The recoveries of the ICA to detect FB1 in maize samples ranged from 91.4 to 118.2%. A quantitative comparison of the fluorescence-based ICA and HPLC-MS/MS analysis of naturally contaminated maize samples indicated good agreement between the two methods (r(2) = 0.93). By replacing the target of interest, the FM-based ICA can easily be extended to other chemical contaminants and thus represents a versatile strategy for food safety analysis.

A total of 420 samples were collected from agrarian households. Whereas 51% (215/420) of the samples were contaminated with one or more toxins, the contamination rates for maize, peanut, and cassava products were 74, 62, and 24%, respectively. The fumonisins (20-5412 μg/kg), aflatoxin B1 (6-645 μg/kg), roquefortine C (1-181 μg/kg), and deoxynivalenol (27-3842 μg/kg) were the most prevalent contaminants in maize. For peanut samples, aflatoxin B1 (6-125 μg/kg) and ochratoxin A (0.3-12 μg/kg) were the main contaminants, whereas aflatoxin B1 (6-194 μg/kg) and penicillic acid (25-184 μg/kg) were detected in the cassava products. Exposures calculated through maize intake for fumonisin B1 and aflatoxin B1 were several-fold higher (2-5 for fumonisin B1 and 10(4)-10(5) for aflatoxin B1) than the health-based guidance values of 2 μg/kg bw/day and 0.15 ng/kg bw/day, respectively. The study design constitutes a good model that can be implemented in other sub-Saharan African countries.

Fumonisin B₁ (FB₁) is a mycotoxin, mainly produced by Fusarium fungi and present in food and feed. It causes harmful effects on human and animal health. Therefore, it is necessary to develop sensitive and reliable screening methods. In this study, a highly sensitive monoclonal antibody (MAb) against FB₁, clone 2D7, was produced, and the 50% inhibition concentration (IC₅₀) of the MAb was 2.2 ng/mL in buffer. The MAb showed high cross-reactivity with fumonisin B₂ (FB₂), and negligible cross-reactivity with other mycotoxins. A sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this MAb was developed for the determination of FB₁ in maize. In spiked samples (100, 200 and 500 μg/kg), the average recoveries ranged from 78 ± 11 to 107 ± 4%, and the coefficient of variation ranged from 3 to 15%. The limit of detection of the icELISA was 5.4 μg/kg. This method was compared to liquid chromatography tandem mass spectrometry (LC-MS/MS) using naturally contaminated samples, and the correlation coefficient was above 0.82. These results show the reliability of the icELISA method for the determination of FB₁ in maize., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This study was designed to develop a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples with particular focus on the optimization of the sample preparation protocol and method validation. All 25 mycotoxins were extracted in a single step with a mixture of methanol/ethyl acetate/water (70:20:10, v/v/v). The method limits of quantification (LOQ) varied from 0.3 μg/kg to 106 μg/kg. Good precision and linearity were observed for most of the mycotoxins. The method was applied for the analysis of naturally contaminated peanut cake, cassava flour and maize samples from the Republic of Benin. All samples analyzed (fifteen peanut cakes, four maize flour and four cassava flour samples) tested positive for one or more mycotoxins. Aflatoxins (total aflatoxins; 10-346 μg/kg) and ochratoxin A (

In the present work, the development of imprinted polymers selective towards mycophenolic acid and their application in food analysis are reported for the first time. To synthesize the molecularly imprinted polymer (MIP) 4-vinylpyridine and ethyleneglycol dimethacrylate were applied as functional monomer and cross-linker, respectively. Besides the toxin itself, the implementation of structural analogues as templates was evaluated. A molecularly imprinted solid-phase extraction (MISPE) procedure was designed for the selective clean-up of maize extracts. Binding experiments and Scatchard analysis indicated the presence of specific binding sites in the imprinted polymers. The imprinting effect varied along with the selected template. The dissociation constant (K(D)) of the higher affinity binding sites ranged from 0.8 μmol/l to 15.6 μmol/l, while the K(D) of the lower affinity binding sites was in the range of 138.5-519.3 μmol/l. The performance of the MIPs throughout the clean-up of spiked maize sample extracts was evaluated and compared with the results obtained when applying a non-imprinted polymer. Depending on the polymers and the spiked concentration, recoveries after MISPE and non-imprinted solid-phase extraction varied respectively from 49% to 84% and from 28% to 31%. The imprinted polymers were superior regarding matrix effect, limit of detection (LOD) and limit of quantification (LOQ). LOD ranged from 0.17 μg/kg to 0.25 μg/kg and LOQ varied from 0.57 μg/kg to 0.82 μg/kg. Analysis of 15 maize samples by liquid chromatography tandem mass spectrometry revealed that the MIPs could be excellent sorbents for clean-up of contaminated food samples., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An interlaboratory validation study was conducted to establish the method performance characteristics of an immunoaffinity column (IAC) cleanup procedure followed by LC/MS for the determination of fumonisins B1 (FB1) and B2 (FB2) and combined FB1 + FB2 in corn. The test portion is extracted with acetonitrile-methanol-water (25 + 25 + 50). The extract is filtered, diluted with phosphate-buffered saline solution, and applied to an IAC. FB1 and FB2 are removed with methanol, followed by water, then directly determined by RPLC with MS detection using selected-ion monitoring of two characteristic ions in each case. Naturally contaminated corn samples were milled to a fine powder and mixed to produce three samples with target levels of combined FB1 + FB2 ranging from 350 to 4000 microg/kg. Of 15 initially participating laboratories, two failed to report results and another did not follow the prescribed method. Thus, valid results were obtained from 12 participants located in 11 countries. Statistical analysis of the results produced RSDr values of 4.6-11.9, 1.9-12.6, and 1.4-11.5% for FB1, FB2, and combined FB1 + FB2, respectively; the corresponding RSDR values were 19.8-23.8, 18.2-25.5, and 18.8-23.2%. The three concentration levels of combined FB1 + FB2 were 534, 1194, and 1954 microg/kg. HorRat values for r and R were all < 2.0, indicating that the method is suitable as a regulatory method for the enforcement of European Union limits for fumonisins in corn.

A molecularly imprinted polymer (MIP) for the recognition of fumonisin B analogues (FB) using 2-(diethylamino) ethyl methacrylate (DEAEM) as functional monomer and trimethylolpropane trimethacrylate (TRIM) as cross-linker was prepared by bulk polymerization in acetonitrile. Fumonisin B(1) (FB(1)) was used as a template molecule. A molecularly imprinted solid-phase extraction (MISPE) procedure was developed for further application in the analysis of FB. The performance of the MIP throughout the clean-up of spiked bell pepper, rice and corn flake sample extracts was compared with the results obtained when using non-imprinted polymer, C(18), strong anion exchange and immunoaffinity sorbents. Extracts were analysed for FB with liquid chromatography-tandem mass spectrometry (LC-MS/MS) after clean-up. Depending on the food matrix and the concentration range of the fumonisins, recoveries after MISPE varied from 62 to 86%, from 62 to 83%, and from 67 to 81% for fumonisin B(1) (FB(1)), fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)), respectively. The selectivity of the synthesized MIP for mycotoxins belonging to the group of FB was confirmed by evaluating cross-reactivity from analogue structures and other mycotoxins. Analysis of 39 naturally contaminated samples (corn flakes) by liquid chromatography tandem mass spectrometry indicated that the synthesized MIP could be an excellent alternative for clean-up and pre-concentration of FB in food samples. Pearson correlations between immunoaffinity clean-up and MISPE were calculated and amounted to 0.923 for FB(1), 0.808 for FB(2), and 0.759 for FB(3). It was shown that the developed MIP could be reused more than 50 times. The synthesis of an FB(1) imprinted polymer and its application in food analysis is reported for the first time.

Two consecutive experiments were performed to evaluate the effects on the immune response of corn cob mix (CCM) in an organic pig diet. The immunoglobulin (Ig) M, IgA and IgG responses against an intramuscularly injected model antigen, bovine thyroglobulin, were used as indicator. The experiments were performed in an organic barn with nine pens of four crossbred pigs (two barrows and two sows) from 45 kg to slaughter. In the first experiment, the organic concentrate was mixed with organic CCM-silage to obtain three concentrate: CCM ratios of 100:0, 80:20 and 60:40 (w:w). In the second experiment, three concentrates were produced to obtain diets with equal nutrient levels on a dry matter basis after 0%, 20% and 40% CCM inclusion. Higher inclusion rates of CCM in the ration were accompanied by lower thyroglobulin-specific IgG responses. These effects could not be attributed to one specific component of the CCM, such as fatty acid composition, although there was a degree of correlation with lower vitamin A concentrations. Mycotoxin concentrations were absent or minimal. The study indicated that dietary ingredient composition may affect immunocompetence.

A total of 205 cornflake samples collected in Belgian retail stores during 2003-2004 were surveyed for the natural occurrence of fumonisin B1 (FB1), B2 (FB2), and B3 (FB3). These cornflake samples, originating from conventional as well as from organic production, were analyzed using an intralaboratory-validated LC-MS/MS method. Additionally, 90 cornflake samples were subjected to rapid screening using a flow-through enzyme immunoassay method to demonstrate the practicability of a screening test coupled to a validated confirmatory LC-MS/MS method for the management of food safety risks. FB(1) concentrations ranged from not detected (nd) [LOD (FB1) = 20 microg/kg] to 464 microg/kg with mean and median concentrations of respectively 104 +/- 113 and 54 microg/kg. For FB2 and FB3, the concentration ranges varied respectively from nd [LOD (FB2) = 7.5 microg/kg] to 43 microg/kg and from nd [LOD (FB3) = 12.5 microg/kg] to 90 microg/kg. Mean concentrations for FB2 and FB3 were respectively 12 +/- 8 and 21 +/- 15 microg/kg, while the median concentration was 11 microg/kg for FB2 and 19 microg/kg for FB3. From the statistical tests (chi2 and ANOVA model III), it could be concluded that the agricultural practice did not have any significant effect on the fumonisin concentrations but that the variation between different batches was significant (p < 0.0001).

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of fumonisin B1 (FB1), B2 (FB2) and B3 (FB3) in cornflakes is described. During method development, special attention was paid to the selection of a suitable internal standard (IS) in order to offer a good alternative for deuterated FB1. In this respect, the C12-sphinganine analogue (2S,3R)-2-aminododecane-1,3-diol was chosen because of its structural similarity to the fumonisin backbone and its chromatographic elution between the target analytes. For the extraction of the fumonisins from the cornflakes matrix, MeOH/H2O (adjusted to pH 4 with 0.1 M HCl; 70:30, v/v), ACN/MeOH/H(2)O (25:25:50, v/v/v) and acidified ACN/MeOH/H2O (25:25:50, v/v/v; pH 4) were evaluated. Preference was given to acidified MeOH/H2O (70:30, v/v) with mean recoveries (n=12) for FB1, FB2 and FB3 of, respectively, 84+/-10, 78+/-7 and 85+/-9%. Cleanup was performed using immunoaffinity columns (FumoniTest, VICAM). The chromatography was performed under isocratic conditions at a flow of 0.3 mL min-1 with a mobile phase consisting of ACN/H2O (60:40, v/v) containing 0.3% formic acid. The mass spectrometer was operated in the positive electrospray ionization (ESI+) mode using multiple reaction monitoring (MRM). An intralaboratory validation was conducted with fortified samples determining limits of detection (LOD), limits of quantification (LOQ), precision, trueness, specificity and measurement uncertainty. The LOD concentrations for FB1, FB2 and FB3 were 20, 7.5 and 12.5 microg/kg. The LOQs were 40 microg/kg for FB1, 15 microg/kg for FB2 and 25 microg/kg for FB3. The coefficients of variation (CVs) under repeatability conditions varied from 11 to 13% for FB1, from 9 to 14% for FB2 and from 7 to 10% for FB3. Under within-laboratory reproducibility conditions, the CVs ranged from 12 to 17% for FB1, from 9 to 16% for FB2 and from 7 to 13% for FB3. The percent bias for FB1 varied from -12 to -10%, while for FB2 and FB3 bias ranged, respectively, from -4 to -2% and from -12 to -5%. The expanded measurement uncertainties for FB1, FB2 and FB3 were, respectively, 19, 18 and 22%., (Copyright (c) 2005 John Wiley & Sons, Ltd.)