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A method based on a hazard analysis critical control point (HACCP)-based standard operating procedure (SOP) has been developed and implemented in community-based (CB) production of complementary foods (CFs) in agrarian regions in rural Ethiopia. The objective of this study was to validate the method in another setup, more specifically in cash crop (coffee and khat) producing communities in Southern Ethiopia. A quasi-experimental study was employed for the method validation. Two villages in the Gedeo zone, Southern Ethiopia were selected and a total of 40 mother-child pairs, 20 in each village, were involved in the study. Sociodemographic data, knowledge, and complementary feeding practices were collected using pretested questionnaires. The training was given to each mother for 1 h about community-based CFs production and implementation of the method. Then, 30 CF samples were collected and the level of aflatoxins (AFB1, AFB2, AFG1, and AFG2) was determined using a validated method. The majority (77.5%) of the mothers understand the importance of complementary feeding and give it to their infants beyond 6 months. Nearly two-thirds (62.5%) of the mothers had knowledge about the health impacts of aflatoxins, but the remaining 37.5% lacked awareness about the health risks of aflatoxins. Mothers were very well-motivated for the training and they promised to implement the knowledge and skill gained from the training for improving the nutritional status of their children. Overall, 23.3% of the CFs were contaminated by aflatoxins. AFB1 and AFG1 were detected in 6.7 and 16.7% of the CF samples, respectively. In cash crop-producing communities around the country, the HACCP-based SOP is easily validated in CB CFs production. Therefore, a scaleup of the method at the national level and beyond is recommended.

Deoxynivalenol (DON) is a type B trichothecene mycotoxin with worldwide high incidence in feed which is produced by Fusarium species. Strategies are needed to eliminate its health risk for livestock and to minimise its economic impact on production. In order to assess the efficacy of potential physical, chemical and biological DON detoxifying agents, a good in vitro model is necessary to perform a fast and high-throughput screening of new compounds before in vivo trials are set up. In this paper, an in vitro model was developed to screen potential commercial products for DON degradation and detoxification. Contaminated feed with potential detoxifying agents are first applied to a simulated gastrointestinal tract (GIT) of a pig, after which detoxification is assessed through a robust, inexpensive and readily applicable Lemna minor L. aquatic plant bioassay which enables evaluation of the residual toxicity of possible metabolites formed by DON detoxifying agents. The GIT simulation enables taking matrix and incubation parameters into account as they can affect the binding, removal or degradation of DON. One product could reduce DON in feed in the GIT model for almost 100% after 6 h. DON metabolites were tentatively identified with LC-MS/MS. This GIT simulation coupled to a detoxification bioassay is a valuable model for in vitro screening and assessing compounds for DON detoxification, and could be expanded towards other mycotoxins.

This research aimed at evaluating the safety, and the type, level and prevalence of mycotoxins in grain sorghum of four sub-Saharan African (SSA) countries (Burkina Faso, Ethiopia, Mali and Sudan). A multi-analyte LC-MS/MS method for quantification of 23 mycotoxins (nivalenol, deoxynivalenol, fusarenon X, neosolaniol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, diacetoxyscirpenol, roquefortine C, HT-2 toxin, alternariol, T-2 toxin, FB1, FB2, FB3, zearalenone, aflatoxin G 1 , aflatoxin G 2 , aflatoxin B 1 , aflatoxin B 2 , sterigmatocystin, OTA, altenuene, alternariol monomethylether) was applied to different sorghum matrices. Of the 1533 analysed samples, 33% were contaminated with at least one of the following mycotoxins: aflatoxins, fumonisins, sterigmatocystin, Alternaria toxins, OTA and zearalenone. Country of origin, colour, source and collection period of sorghum samples significantly influenced the type, level and prevalence of mycotoxins. Sterigmatocystin (15%), fumonisins (17%) and aflatoxins (13%) were the most prevalent. FB1 (274 ± 585 µg/kg) had the highest mean concentration followed by FB2 (214 ± 308 µg/kg) while diacetoxyscirpenol (8.12 ± 19.2 µg/kg) and HT-2 (11.9 ± 0.00 µg/kg) had the lowest concentrations. Neosolaniol, fusarenon-X, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, T-2 toxin, nivalenol and roquefortine C were not detected in any of the samples. Sudan had the lowest prevalence and mean concentration of all mycotoxins. Pink sorghum had the highest concentrations of fumonisins and aflatoxins. Mycotoxins from Aspergillus spp. and Alternaria spp. are the mycotoxins of concern in SSA grain sorghum with regard to prevalence, concentration and possible health risk from exposure. Based on the performed risk characterisation, daily consumption of sorghum containing aflatoxins, alternariol, alternariol monomethyl ether, sterigmatocystin and OTA could result in exceeding the established health-based guidance values for these toxins.

The direct measurement of biomarkers of exposure in biological fluids such as urine has become important for assessing aflatoxin exposure in humans as it is the only tool that integrates exposures from various routes. For this reason, a study was conducted to assess aflatoxin exposure among young children in Ethiopia using urinary biomarkers. A cross-sectional study was conducted in ten Woredas (Districts) from Amhara and Tigray regional states of Ethiopia including 200 children (aged 1-4 years). A total of 200 urine samples were collected from 200 children and assessed for the levels of aflatoxin B 1 (AFB 1 ), aflatoxin B 2 (AFB 2 ), aflatoxin G 1 (AFG 1 ), aflatoxin G 2 (AFG 2 ) and aflatoxin M 1 (AFM 1 ) using a validated LC-MS/MS method. Aflatoxins were detected in 34/200 (17%) of the urine samples whereby four out of five analysed aflatoxins were detected. AFM 1 was detected in 14/200 (7%) of the urine samples in a range of 0.06-0.07 ng/mL. AFB 2 , AFG 2 and AFG 1 were detected in respectively 9/200 (4.5%), 6/200 (3%) and 5/200 (2.5%) of the urine samples whereas AFB 1 was not detected in any of the samples. In this study, there was no association between the different malnutrition categories (stunted, wasting and underweight) and aflatoxin exposure. However, the biomarker analysis showed a clear exposure of young children to aflatoxins. Therefore, awareness to the public is important to prevent potential health consequences of aflatoxins.

The fungus Aspergillus flavus is known for its ability to produce the toxic and carcinogenic aflatoxins in food and feed. While aflatoxins are of most concern, A. flavus is predicted to be capable of producing many more metabolites based on a study of its complete genome sequence. Some of these metabolites could be of great importance in food and feed safety. Here we describe an analytical methodology based on Orbitrap HRMS technology that allows the untargeted determination of fungal metabolites, in support of the study of the function of genes involved in secondary metabolism in fungi. The applied strategy implies the detection and identification of differentially expressed metabolites in extracts of wild-type and mutant fungal strains, using Orbitrap high-resolution mass spectrometry (HRMS) accurate mass data. The suitability of this approach was demonstrated by the confirmation of previously characterised genes involved in the aflatoxin biosynthetic pathway, namely a polyketide synthase (pksA), an oxidoreductase (ordA) and a methyltransferase (omtA) gene. Subsequently, the proposed methodology was applied for the detection and identification of metabolites produced by a yet uncharacterised gene cluster in A. favus, cluster 23. Comparative Orbitrap HRMS analysis of extracts of A. flavus wild-type strain and an over-expression mutant for the transcription factor of gene cluster 23 (lepE) demonstrated that this gene cluster is responsible for the production a set of 2-pyridone derivatives, the leporins. Besides the known derivatives leporin B and leporin B precursor that could be identified by automatic de-replication of the accurate mass data, five other compounds belonging to this class of fungal secondary metabolites were detected and identified for the first time, combining MS and multiple-stage MS data.

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.

The filamentous fungus Aspergillus flavus is one of the most important species in the Aspergillus genus and is distributed worldwide as a prevalent aflatoxin-producing food and feed contaminant. A. flavus contains more than 55 gene clusters that are predicted to encode proteins involved in secondary metabolite production. One of these, cluster 27, contains a polyketide synthase (pks27) gene that encodes a protein that is highly homologous to the aflatoxin cluster PKS. Comparative metabolomics, using ultra-high performance liquid chromatography (UHPLC) coupled to high resolution Orbitrap mass spectrometry (MS) was used to detect metabolites differentially expressed in the A. flavus wild-type and ∆pks27 mutant strains. Metabolite profiling was aided by a statistical differential analysis of MS data using SIEVE software. This differential analysis combined with accurate mass data from the Orbitrap and ion trap multiple stage MS allowed four metabolites to be identified that were produced only by the wild-type culture. These included asparasone A (358 Da), an anthraquinone pigment, and related anthraquinones with masses of 316, 340 and 374 Da. These latter three compounds had similar fragmentation patterns to that of asparasone A. The 316 Da anthraquinone is particularly interesting because it is most likely formed by incorporation of seven malonyl-CoA units rather than the eight units required for the formation of asparasone A. The 340 and 374 Da metabolites are the dehydration and an oxy-derivative of asparasone A, respectively. Asparasone A was also identified in extracts from several other Aspergillus species.

Mycotoxins are harmful food contaminants. Currently, human exposure assessment to these toxins is often based on calculations combining mycotoxin occurrence data in food with population data on food consumption. Because of limitations inherent to that approach, biomarkers have been proposed as a suitable alternative whereby a more accurate assessment of exposure at the individual level can be performed. The BIOMYCO study is designed to assess human mycotoxin exposure using urinary biomarkers of exposure. Over the different seasons of 2013 and 2014, morning urine is gathered in a representative part of the Belgian population according to a designed study protocol, whereby 140 children (3-12 years old) and 278 adults (19-65 years old) are selected based on random cluster sampling stratified for sex, age and geographical areas. Every participant completes a food frequency questionnaire to assess the consumption of relevant foodstuffs (n = 43) of both the day before the urine collection and the previous month. Validated multi-toxin LC-MS/MS methods are used to analyse aflatoxins, fumonisins, ochratoxin A, trichothecenes, zearalenone and their metabolites in morning urine. The study protocol is approved by the ethical committee of the Ghent University Hospital. Within this paper, study design and methods are described. The BIOMYCO study is the first study whereby a multi-toxin approach is applied for mycotoxin exposure assessment in adults and children on a large scale. Moreover, it is the first study that will describe the exposure to an elaborated set of mycotoxins in the Belgian population. In first instance, descriptive analysis will be performed, describing the exposure to mycotoxins for the child and adult group. Exposure of different subgroups will be compared. Furthermore, correlations between the mycotoxin concentrations measured and the food consumption reported will be estimated to explore whether the mycotoxin exposure could be explained by the consumption of certain foods.

An immunogen synthesis strategy was designed to develop anti-deoxynivalenol (DON) monoclonal antibodies with low cross-reactivity against structurally similar trichothecenes. A total of eight different DON immunogens were synthesised, differing in the type and position of the linker on the DON molecule. After immunisation, antisera from mice immunised with different DON immunogens were checked for the presence of relevant antibodies. Then, both homologous and heterologous enzyme-linked immunosorbent assays (ELISAs) were performed for hybridoma screening. Finally, three monoclonal antibodies against DON and its analogues were generated. In addition, monoclonal antibody 13H1 could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyldeoxynivalenol (15-ADON) > DON, with IC₅₀ ranging from 1.14 to 2.13 µg ml⁻¹. Another monoclonal antibody 10H10 manifested relatively close sensitivities to DON, 3-acetyldeoxynivalenol (3-ADON) and 15-ADON, with IC₅₀ values of 22, 15 and 34 ng ml⁻¹, respectively. Using an indirect ELISA format decreases the 10H10 sensitivity to 15-ADON with 92%. A third monoclonal antibody 2A9 showed to be very specific and sensitive to 3-ADON, with IC₅₀ of 0.38 ng ml⁻¹. Using both 2A9 and 10H10 monoclonal antibodies allows determining sole DON contamination.

An LC-MS/MS method was developed and validated for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin, HT-2-toxin and metabolites, including 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deoxynivalenol-3-glucoside, α-zearalenol, β-zearalenol, zearalenone-4-glucoside, α-zearalenol-4-glucoside, β-zearalenol-4-glucoside and zearalenone-4-sulfate in maize, wheat, oats, cornflakes and bread. Extraction was performed with acetonitrile/water/acetic acid (79/20/1, v/v/v) followed by a hexane defatting step. After filtration, the extract was evaporated and the residue was redissolved in mobile phase for injection. The mobile phase, which consisted of a mixture of methanol and water with 10 mM ammonium acetate, was adjusted to pH 3 with glacial acetic acid. A sample clean-up procedure was not included because of the low recoveries of free and masked mycotoxins and their differences in polarity. The method allowed the simultaneous determination of 13 Fusarium mycotoxins in a one-step chromatographic run using a Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer. The method was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, expanded measurement uncertainty and specificity. The limits of detection varied from 5 to 13 ng g⁻¹; those for the limit of quantification from 10 to 26 ng g⁻¹. The results of the performance characteristics of the developed LC-MS/MS method were in good agreement with the criteria mentioned in Commission Regulation (EC) No. 401/2006. Thirty samples of a variety of food and feed matrices were sampled and analysed between July 2010 and January 2011.

Seven commercially available deoxynivalenol (DON) and zearalenone (ZEN) immunoaffinity columns (IACs) were tested for cross-reactivity to conjugated forms (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, DON-3-glucoside, DON-3-glucuronide, ZEN-glucosides, ZEN-glucuronide) and metabolites (de-epoxydeoxynivalenol, α-zearalenol, β-zearalenol) and nivalenol (NIV), using a semi-quantitative multi-mycotoxin ultra-performance liquid chromatography-tandem mass spectrometry method. The DON IACs showed cross-reactivity for nearly all DON derivatives tested. The ZEN IACs showed limited cross-reactivity to some of the ZEN derivatives. The IACs were evaluated for their potential use as sample clean-up for mycotoxins in serum., (© 2011 Taylor & Francis)

In vitro risk assessment of dietary contaminants has become a priority in human food safety. This paper proposes an in vitro approach associating different complementary tools in an original toolbox and aims to improve the assessment of the toxicological impact of dietary contaminants at realistic human exposure levels, with a special focus on the intestinal compartment. The system is based on the use of four complementary cellular tools, namely stress gene induction in transgenic strains of Escherichia coli, modulation of the activity of key biotransformation enzymes (cytochrome P-450 (CYP) 1A1 and 3A4) in a human intestinal cell line, and activation of aryl hydrocarbon receptor (AhR) and oestrogenic receptor (ER)-dependent genes in agonistic and antagonistic assays with luciferase reporter cells. It was applied to four chosen model molecules: ochratoxin A (OTA) and deoxynivalenol (DON), two common food-borne mycotoxins, and imazalil (IMA) and benomyl (BEN), two fungicides widely occurring in foodstuffs. All these assays were performed at or around a realistic intestinal concentration, determined through a deterministic approach based on the calculation of a theoretical maximum daily intake (TMDI). Using the four model molecules, it is clearly highlighted that induction of CYP1A1 activity and inhibition of CYP3A4 activity occurred in Caco-2 cells at a realistic intestinal concentration of IMA. Furthermore, some bacterial stress genes were induced in a range of realistic concentrations, following exposure to DON and IMA. In addition, BEN clearly provoked an ER agonistic activity in a human oestrogen sensitive reporter cell line. All these results are in accordance with the literature, suggesting that the in vitro toolbox constitutes an interesting approach in order to obtain a first 'fingerprint' of dietary contaminants at realistic human exposure for further risk assessment., (© 2011 Taylor & Francis)

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.

A multi-analyte method for the liquid chromatography-tandem mass spectrometric determination of mycotoxins in food supplements is presented. The analytes included A and B trichothecenes (nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin and T-2 toxin), aflatoxins (aflatoxin-B(1), aflatoxin-B(2), aflatoxin-G(1) and aflatoxin-G(2)), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin-B(1), fumonisin-B(2) and fumonisin-B(3)), ochratoxin A, zearalenone, beauvericin and sterigmatocystin. Optimization of the simultaneous extraction of these toxins and the sample pretreatment procedure, as well as method validation were performed on maca (Lepidium meyenii) food supplements. The results indicated that the solvent mixture ethyl acetate/formic acid (95:5, v/v) was the best compromise for the extraction of the analytes from food supplements. Liquid-liquid partition with n-hexane was applied as partial clean-up step to remove excess of co-extracted non-polar components. Further clean-up was performed on Oasis HLB cartridges. Samples were analysed using an Acquity UPLC system coupled to a Micromass Quattro Micro triple quadrupole mass spectrometer equipped with an electrospray interface operated in the positive-ion mode. Limits of detection and quantification were in the range of 0.3-30 ng g(-1) and 1-100 ng g(-1), respectively. Recovery yields were above 60% for most of the analytes, except for nivalenol, sterigmatocystine and the fumonisins. The method showed good precision and trueness. Analysis of different food supplements such as soy (Glycine max) isoflavones, St John's wort (Hypericum perforatum), garlic (Allium sativum), Ginkgo biloba, and black radish (Raphanus niger) demonstrated the general applicability of the method. Due to different matrix effects observed in different food supplement samples, the standard addition approach was applied to perform correct quantitative analysis. In 56 out of 62 samples analysed, none of the 23 mycotoxins investigated was detected. Positive samples contained at least one of the toxins fumonisin-B(1), fumonisin-B(2), fumonisin-B(3) and ochratoxin A.

Different cheese samples produced in Latvia (eight) and Belgium (13) were analysed for their sterigmatocystin (STC) content. Only two (9.5%) of the samples were positive for STC with concentration levels of 1.23 and 0.52 microg kg(-1), respectively. Five (24%) samples contained STC above the limit of detection (0.03 microg kg(-1)) but below the limit of quantification (0.1 microg kg(-1)), A sensitive liquid chromatography-electrospray positive ionisation-tandem mass spectrometry (LC-MS/MS) method, which was previously developed for the analysis of STC in grains, was modified and applied to the analysis of STC in cheese. This method involved sample extraction with acetonitrile-water (90 : 10, v/v), defatting with n-hexane, solid-phase extraction, separation on a reversed-phase C(18) column, and STC detection by LC-MS/MS.