Carbon dioxide production as an indicator of Aspergillus flavus colonisation and aflatoxins/cyclopiazonic acid contamination in shelled peanuts stored under different interacting abiotic factors.

Aspergillus flavus is the main xerophylic species colonising stored peanuts resulting in contamination with aflatoxins (AFs) and cyclopiazonic acid (CPA). This study evaluated the relationship between storage of shelled peanuts under interacting abiotic conditions on (a) temporal respiration (R) and cumulative CO2 production, (b) dry matter losses (DMLs) and (c) aflatoxin B1 (AFB1) and CPA accumulation. Both naturally contaminated peanuts and those inoculated with A. flavus were stored for 7-days under different water activities (aw; 0.77-0.95) and temperatures (20-35°C). There was an increase in the temporal CO2 production rates in wetter and warmer conditions, with the highest respiration at 0.95 aw + A. flavus inoculum at 30°C (2474 mg CO2kg-1h-1). The DMLs were modelled to produce contour maps of the environmental conditions resulting in maximum/minimum losses. Maximum mycotoxin contamination was always at 0.95 aw although optimal temperatures were 25-30°C for AFs and 30-35°C for CPA. These results showed a correlation between CO2 production and mycotoxin accumulation. They also provide valuable information for the creation of a database focused on the development of a post-harvest decision support system to determine the relative risks of contamination with these mycotoxins in stored shelled peanuts.

Influence of storage environment on maize grain: CO2 production, dry matter losses and aflatoxins contamination.

Poor storage of cereals, such as maize can lead to both nutritional losses and mycotoxin contamination. The aim of this study was to examine the respiration of maize either naturally contaminated or inoculated with Aspergillus flavus to examine whether this might be an early and sensitive indicator of aflatoxin (AF) contamination and relative storability risk. We thus examined the relationship between different interacting storage environmental conditions (0.80-0.99 water activity (aw) and 15-35°C) in naturally contaminated and irradiated maize grain + A. flavus on relative respiration rates (R), dry matter losses (DMLs) and aflatoxin B1 and B2 (AFB1-B2) contamination. Temporal respiration and total CO2 production were analysed by GC-TCD, and results used to calculate the DMLs due to colonisation. AFs contamination was quantified at the end of the storage period by HPLC MS/MS. The highest respiration rates occurred at 0.95 aw and 30-35°C representing between 0.5% and 18% DMLs. Optimum AFs contamination was at the same aw at 30°C. Highest AFs contamination occurred in maize colonised only by A. flavus. A significant positive correlation between % DMLs and AFB1 contamination was obtained (r = 0.866, p < 0.001) in the irradiated maize treatments inoculated with A. flavus. In naturally contaminated maize + A. flavus inoculum loss of only 0.56% DML resulted in AFB1 contamination levels exceeding the EU legislative limits for food. This suggests that there is a very low threshold tolerance during storage of maize to minimise AFB1 contamination. This data can be used to develop models that can be effectively used in enhancing management for storage of maize to minimise risks of mycotoxin contamination.

Mycotoxin contamination of foods in Southern Africa: A 10-year review (2007-2016).

Major staple foods in Southern Africa are prone to mycotoxin contamination, posing health risks to consumers and consequent economic losses. Regional climatic zones favor the growth of one or more main mycotoxin producing fungi, Aspergillus, Fusarium and Penicillium. Aflatoxin contamination is mainly reported in maize, peanuts and their products, fumonisin contamination in maize and maize products and patulin in apple juice. Lack of awareness of occurrence and risks of mycotoxins, poor agricultural practices and undiversified diets predispose populations to dietary mycotoxin exposure. Due to a scarcity of reports in Southern Africa, reviews on mycotoxin contamination of foods in Africa have mainly focused on Central, Eastern and Western Africa. However, over the last decade, a substantial number of reports of dietary mycotoxins in South Africa have been documented, with fewer reports documented in Botswana, Lesotho, Malawi, Mozambique, Zambia and Zimbabwe. Despite the reported high dietary levels of mycotoxins, legislation for their control is absent in most countries in the region. This review presents an up-to-date documentation of the epidemiology of mycotoxins in agricultural food commodities and discusses the implications on public health, current and recommended mitigation strategies, legislation, and challenges of mycotoxin research in Southern Africa.

Newly discovered ergot alkaloids in Sorghum ergot Claviceps africana occurring for the first time in Israel.

Sorghum ergot is a disease caused commonly by C. africana. In 2015, ergot was identified for the first time in sorghum fields in Israel, leading to measures of eradication and quarantine. The aims of the study were to identify the ergot species by molecular and ergot alkaloid profile analysis, to determine the ergot alkaloid profile in pure honeydew and in infected sorghum silages and to estimate the safety of sorghum silages as a feed source. C. africana was rapidly and reliably identified by microscopical and molecular analysis. Dihydroergosine was identified as the major ergot alkaloid. Dihydrolysergol and dihydroergotamine were identified for the first time as significant ergot alkaloid components within the C. africana sclerotia, thereby providing for the first time a proof for the natural occurrence of dihydroergotamine. The sorghum silages were found to be safe for feed consumption, since the ergot alkaloids and the regulated mycotoxins were below their regulated limits.

Microbial secondary metabolites in homes in association with moisture damage and asthma.

We aimed to characterize the presence of microbial secondary metabolites in homes and their association with moisture damage, mold, and asthma development. Living room floor dust was analyzed by LC-MS/MS for 333 secondary metabolites from 93 homes of 1-year-old children. Moisture damage was present in 15 living rooms. At 6 years, 8 children had active and 15 lifetime doctor-diagnosed asthma. The median number of different metabolites per house was 17 (range 8-29) and median sum load 65 (4-865) ng/m(2) . Overall 42 different metabolites were detected. The number of metabolites present tended to be higher in homes with mold odor or moisture damage. The higher sum loads and number of metabolites with loads over 10 ng/m(2) were associated with lower prevalence of active asthma at 6 years (aOR 0.06 (95% CI <0.001-0.96) and 0.05 (<0.001-0.56), respectively). None of the individual metabolites, which presence tended (P < 0.2) to be increased by moisture damage or mold, were associated with increased risk of asthma. Microbial secondary metabolites are ubiquitously present in home floor dust. Moisture damage and mold tend to increase their numbers and amount. There was no evidence indicating that the secondary metabolites determined would explain the association between moisture damage, mold, and the development of asthma.

Quantitation of multiple mycotoxins and cyanogenic glucosides in cassava samples from Tanzania and Rwanda by an LC-MS/MS-based multi-toxin method.

A multi-mycotoxin method based on liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used for a mycotoxin survey in 627 samples of processed cassava collected from different districts across Tanzania and Rwanda after the method performance for this matrix had been determined. Matrix effects as well as extraction efficiencies were found to be similar to most other previously investigated matrices with the exception of distinct matrix effects in the negative ionisation mode for early eluting compounds. Limits of detection were far below the regulatory limits set in the European Union for other types of commodities. Relative standard deviations were generally lower than 10% as determined by replicates spiked on two concentration levels. The sample-to-sample variation of the apparent recoveries was determined for 15 individually spiked samples during three different analytical sequences. The related standard deviation was found to be lower than 15% for most of the investigated compounds, thus confirming the applicability of the method for quantitative analysis. The occurrence of regulated mycotoxins was lower than 10% (with the exception of zearalenone) and the related limits were exceeded only in few samples, which suggests that cassava is a comparatively safe commodity as regards mycotoxins. The most prevalent fungal metabolites were emodin, kojic acid, beauvericin, tryptophol, 3-nitropropionic acid, equisetin, alternariol methylether, monocerin, brevianamide F, tenuazonic acid, zearalenone, chrysophanol, monilifomin, enniatins, apicidin and macrosporin. The related concentrations exceeded 1 mg kg(-1) only in few cases. However, extremely high levels of cyanogenic plant toxins, which had been previously added to the method, were observed in few samples, pointing out the need for improved post-harvest management to decrease the levels of these compounds.

Mycotoxins in corn and wheat silage in Israel.

Silage is an important feed source for intensive dairy herds worldwide. Fungal growth and mycotoxin production before and during silage storage is a well-known phenomenon, resulting in reduced nutritional value and a possible risk factor for animal health. With this in mind, a survey was conducted to determine for the first time the occurrence of mycotoxins in corn and wheat silage in Israel. A total of 30 corn and wheat silage samples were collected from many sources and analysed using a multi-mycotoxin method based on LC-MS/MS. Most mycotoxins recorded in the present study have not been reported before in Israel. Overall, 23 mycotoxins were found in corn silage; while wheat silage showed a similar pattern of mycotoxin occurrence comprising 20 mycotoxins. The most common post-harvest mycotoxins produced by the Penicillium roqueforti complex were not found in any tested samples, indicative of high-quality preparation and use of silage. Moreover, none of the European Union-regulated mycotoxins--aflatoxin B1, ochratoxin, T-2 toxin, diacetoxyscirpenol and deoxynivalenol--were found above their limits of detection (LODs). The Alternaria mycotoxins--macrosporin, tentoxin and alternariol methyl ether--were highly prevalent in both corn and wheat silage (>80%), but at low concentrations. The most prominent (>80%) Fusarium mycotoxins in corn silage were fusaric acid, fumonisins, beauvericin, monilifomin, equisetin, zearalenone and enniatins, whereas in wheat silage only beauvericin, zearalenone and enniatins occurred in more than 80% of the samples. The high prevalence and concentration of fusaric acid (mean = 765 µg kg⁻¹) in Israeli corn silage indicates that this may be the toxin of highest potential concern to dairy cow performance. However, more data from different harvest years and seasons are needed in order to establish a more precise evaluation of the mycotoxin burden in Israeli silage.

Fungal and mycotoxin assessment of dried edible mushroom in Nigeria.

In order to determine whether dried mushrooms are a foodstuff that may be less susceptible to infection by toxigenic molds and consequently to mycotoxin contamination, 34 dried market samples were analyzed. Fungal population was determined in the samples by conventional mycological techniques and molecular studies, while the spectrum of microbial metabolites including mycotoxins was analyzed by a liquid chromatography tandem mass spectrometric method covering 320 metabolites. Molds such as Fusarium, Penicillium, Trichoderma and aflatoxigenic species of Aspergillus (Aspergillus flavus and Aspergillus parvisclerotigenus) were recovered from all samples at varying levels. None of the mycotoxins addressed by regulatory limits in the EU was positively identified in the samples. However, 26 other fungal metabolites occurred at sub- to medium µg/kg levels in the samples, including aflatoxin/sterigmatocystin bio-precursors, bis-anthraquinone derivatives from Talaromyces islandicus, emerging toxins (e.g. enniatins) and other Fusarium metabolites, and clavine alkaloids. Although little is known on the toxicology of these substances, the absence of aflatoxins and other primary mycotoxins suggests that dried mushrooms may represent a relatively safe type of food in view of mycotoxin contamination.

Isotopic labeling-assisted metabolomics using LC-MS.

Metabolomics has emerged as the latest of the so-called "omics" disciplines and has great potential to provide deeper understanding of fundamental biochemical processes at the biological system level. Among recent technological developments, LC-HRMS enables determination of hundreds to thousands of metabolites over a wide range of concentrations and has developed into one of the most powerful techniques in non-targeted metabolomics. The analysis of mixtures of in-vivo-stable isotopic-labeled samples or reference substances with un-labeled samples leads to specific LC-MS data patterns which can be systematically exploited in practically all data-processing steps. This includes recognition of true metabolite-derived analytical features in highly complex LC-MS data and characterization of the global biochemical composition of biological samples. In addition, stable-isotopic labeling can be used for more accurate quantification (via internal standardization) and identification of compounds in different organisms.

Mycotoxins and fungal metabolites in groundnut- and maize-based snacks from Nigeria.

This exploratory study was aimed at investigating the spectrum of fungal metabolites in the processed food and snacks. Twenty types of snacks made separately from groundnut (n = 10), maize (n = 8) and a combination of groundnut and maize (n = 2) were analysed for naturally occurring mycotoxins and other fungal metabolites by a liquid chromatography-tandem mass spectrometric multi-mycotoxin method. A total of 18, 21 and 32 metabolites were detected and quantified in the groundnut-, groundnut/maize- and maize-based snacks, respectively. Aflatoxins contaminated 2, 3 and 5 of the groundnut/maize-, groundnut- and maize-based snacks at concentrations up to 14, 1041 and 74 µg kg(-1), respectively. Thus, the National Agency for Food and Drug Administration and Control (NAFDAC) recommended limit of 20 µg kg(-1) for aflatoxins was exceeded in 6 of the 20 snacks. Fumonisins contaminated all the maize- and groundnut/maize-based snacks with higher concentrations in the maize-based snacks (mean = 218.7 µg kg(-1)) compared with the groundnut/maize-based snacks (mean = 178.5 µg kg(-1)). Up to 26 different metabolites were found to co-occur in the same samples, thus posing an additional threat to the consumers due to possible additive and/or synergistic effects.

Fungal and bacterial metabolites in commercial poultry feed from Nigeria.

Metabolites of toxigenic fungi and bacteria occur as natural contaminants (e.g. mycotoxins) in feedstuffs making them unsafe to animals. The multi-toxin profiles in 58 commercial poultry feed samples collected from 19 districts in 17 states of Nigeria were determined by LC/ESI-MS/MS with a single extraction step and no clean-up. Sixty-three (56 fungal and seven bacterial) metabolites were detected with concentrations ranging up to 10,200 µg kg⁻¹ in the case of aurofusarin. Fusarium toxins were the most prevalent group of fungal metabolites, whereas valinomycin occurred in more than 50% of the samples. Twelve non-regulatory fungal and seven bacterial metabolites detected and quantified in this study have never been reported previously in naturally contaminated stored grains or finished feed. Among the regulatory toxins in poultry feed, aflatoxin concentrations in 62% of samples were above 20 µg kg⁻¹, demonstrating high prevalence of unsafe levels of aflatoxins in Nigeria. Deoxynivalenol concentrations exceeded 1000 µg kg⁻¹ in 10.3% of samples. Actions are required to reduce the consequences from regulatory mycotoxins and understand the risks of the single or co-occurrence of non-regulatory metabolites for the benefit of the poultry industry.

Single-kernel analysis of fumonisins and other fungal metabolites in maize from South African subsistence farmers.

Fumonisins are important Fusarium mycotoxins mainly found in maize and derived products. This study analysed maize from five subsistence farmers in the former Transkei region of South Africa. Farmers had sorted kernels into good and mouldy quality. A total of 400 kernels from 10 batches were analysed; of these 100 were visually characterised as uninfected and 300 as infected. Of the 400 kernels, 15% were contaminated with 1.84-1428 mg kg(-1) fumonisins, and 4% (n=15) had a fumonisin content above 100 mg kg(-1). None of the visually uninfected maize had detectable amounts of fumonisins. The total fumonisin concentration was 0.28-1.1 mg kg(-1) for good-quality batches and 0.03-6.2 mg kg(-1) for mouldy-quality batches. The high fumonisin content in the batches was apparently caused by a small number (4%) of highly contaminated kernels, and removal of these reduced the average fumonisin content by 71%. Of the 400 kernels, 80 were screened for 186 microbial metabolites by liquid chromatography-tandem mass spectrometry, detecting 17 other fungal metabolites, including fusaric acid, equisetin, fusaproliferin, beauvericin, cyclosporins, agroclavine, chanoclavine, rugulosin and emodin. Fusaric acid in samples without fumonisins indicated the possibility of using non-toxinogenic Fusaria as biocontrol agents to reduce fumonisin exposure, as done for Aspergillus flavus. This is the first report of mycotoxin profiling in single naturally infected maize kernels.

Genotyping and phenotyping of Fusarium graminearum isolates from Germany related to their mycotoxin biosynthesis.

Fusarium graminearum is the most important pathogen causing Fusarium head blight (FHB) of small cereal grains worldwide responsible for quantitative and qualitative yield losses. The presence in crops is often associated with mycotoxin contamination of foodstuff limiting its use for human and animal consumption. A collection of isolates of F. graminearum from Germany was characterized genetically and chemically for their potential to produce the B trichothecenes deoxynivalenol (DON) and nivalenol (NIV). Molecular methods with eight PCR assays were implemented based on functional Tri7 and Tri13 genes and on the tri5-tri6 intergenic region to differentiate between chemotaxonomic groups DON and NIV, resulting in a marked majority (61/63) of DON chemotypes. Mycotoxins produced on rice kernels were quantified by means of LC-MSMS including DON, NIV, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON), DON-3-glucoside, fusarenon X, as well as zearalenone; all of them proving to be present in high concentration among the isolates. All DON-chemotype isolates also produced lower amounts of NIV with the amount being positively correlated (R²=0.89) to the DON amount. 15-ADON and 3-ADON are reported to be produced simultaneously by the isolates, the former dominating over the latter in all but one isolate. Fungal biomass, was quantified via ergosterol amount on rice. It was used to calculate specific mycotoxin production per biomass of isolates, ranging from 0.104 to 1.815mg DON mg-1 ergosterol, presenting a Gaussian distribution. Genotype and phenotype characterization revealed discrepancies with respect to mycotoxin production potential of the fungi, i.e. isolates from one chemotype were able to produce mycotoxins from other chemotypes in considerable amounts.

Evaluation of LC-high-resolution FT-Orbitrap MS for the quantification of selected mycotoxins and the simultaneous screening of fungal metabolites in food.

A liquid chromatography-high-resolution mass spectrometry-based method is reported for the quantification of 20 selected mycotoxins and the simultaneous screening for 200 fungal metabolites in food. For regulated mycotoxins, such as aflatoxins, fumonisins, ochratoxin A, zearalenone and trichothecenes, the evaluation of the method performance characteristics, such as precision, trueness, limit of detection and matrix effects, has been exemplified for the matrix maize. In the case of the limit of detection, an alternative evaluation approach for high-resolution FT-Orbitrap data is proposed. Measurements of the signal-to-noise ratios obtained from 'full-profile mode' data led to detection limits between 8 and 160 ng g(-1). Eight naturally contaminated wheat- and maize-based matrix test materials, originating from interlaboratory comparison studies, were used to confirm the trueness of the method for deoxynivalenol, zearalenone, fumonisin B(1) and B(2), HT-2, and T-2 toxin. In addition to accurate quantification of the most relevant mycotoxins, the full-scan chromatograms were used to investigate the potential of the FT-Orbitrap to screen simultaneously for a large number of fungal metabolites. First, a list of 200 metabolites, potentially being present in food samples, was established. Next, specific detection and identification criteria were defined, which are based on accurate mass, peak intensity and isotopologue ratio. The application of these criteria to the suspected metabolites from the list resulted in the putative identification of 13 fungal metabolites in addition to the target toxins.

Co-occurrence of toxic bacterial and fungal secondary metabolites in moisture-damaged indoor environments.

UNLABELLED: Toxic microbial secondary metabolites have been proposed to be related to adverse health effects observed in moisture-damaged buildings. Initial steps in assessing the actual risk include the characterization of the exposure. In our study, we applied a multi-analyte tandem mass spectrometry-based methodology on sample materials of severely moisture-damaged homes, aiming to qualitatively and quantitatively describe the variety of microbial metabolites occurring in building materials and different dust sample types. From 69 indoor samples, all were positive for at least one of the 186 analytes targeted and as many as 33 different microbial metabolites were found. For the first time, the presence of toxic bacterial metabolites and their co-occurrence with mycotoxins were shown for indoor samples. The bacterial compounds monactin, nonactin, staurosporin and valinomycin were exclusively detected in building materials from moist structures, while chloramphenicol was particularly prevalent in house dusts, including settled airborne dust. These bacterial metabolites are highly bioactive compounds produced by Streptomyces spp., a group of microbes that is considered a moisture damage indicator in indoor environments. We show that toxic bacterial metabolites need to be considered as being part of very complex and diverse microbial exposures in 'moldy' buildings. PRACTICAL IMPLICATIONS: Bacterial toxins co-occur with mycotoxins in moisture-damaged indoor environments. These compounds are measurable also in settled airborne dust, indicating that inhalation exposure takes place. In attempts to characterize exposures to microbial metabolites not only mycotoxins but also bacterial metabolites have to be targeted by the analytical methods applied. We recommend including analysis of samples of outdoor air in the course of future indoor assessments, in an effort to better understand the outdoor contribution to the indoor presence of microbial toxins. There is a need for a sound risk assessment concerning the exposure to indoor microbial toxins at concentrations detectable in moisture-damaged indoor environments.

Sampling of cereals and cereal-based foods for the determination of ochratoxin A: an overview.

The mycotoxin ochratoxin A (OTA) is known to be heterogeneously distributed both intrinsically (from one individual food item to the next) as well as distributionally (throughout a sample of individual food items) in cereals and cereal-based foods. Therefore, proper sampling and sample comminution are special challenges, but are prerequisites for obtaining sound analytical data. This paper outlines the issue of the sampling process for cereals and cereal-based foods, starting with the planning phase, followed by the sampling step itself and the formation of analytical samples. The sampling of whole grain and retail-level cereal-based foods will be discussed. Furthermore, possibilities to reduce sampling variance are presented.

Occurrence of free and conjugated Fusarium mycotoxins in cereal-based food.

A collection of 84 cereal-based food products in 25 composites, including beer, was screened for the presence of deoxynivalenol, zearalenone, and their respective metabolites deoxynivalenol-3-glucopyranoside, 3-acetyl-deoxynivalenol, zearalenol-4-glucopyranoside, alpha-zearalenol, beta-zearalenol, alpha-zearalenol-4-glucopyranoside, beta-zearalenol-4-glucopyranoside, and zearalenone-4-sulfate. The most abundant analyte was zearalenone-4-sulfate, which was found in 13 composites, albeit in low concentrations. Furthermore, deoxynivalenol was detected in eight, zearalenone in seven, and deoxynivalenol-3-glucopyranoside in two composites. None of the remaining six analytes was found in any matrices, which suggests that, if at all present, the concentrations of these latter metabolites are very low and, hence, do not impose any danger to consumers. The highest mycotoxin content was found in bran flakes with 254 ng g(-1) deoxynivalenol, 6 ng g(-1) zearalenone-4-sulfate, and 44 ng g(-1) zearalenone.

Preparation and characterization of the conjugated Fusarium mycotoxins zearalenone-4O-beta-D-glucopyranoside, alpha-zearalenol-4O-beta-D-glucopyranoside and beta-zearalenol-4O-beta-D-glucopyranoside by MS/MS and two-dimensional NMR.

Glucosides of several Fusarium mycotoxins occur in naturally infected cereals and may contribute to an increased content to the total mycotoxin load of food and feed. The paper presents the results of a fermentation procedure to produce zearalenone-4O-beta-D-glucopyranoside from zearalenone using an engineered Saccharomyces cerevisiae strain, expressing the Arabidopsis thaliana UDP-glucosyltransferase UGT73C6. About 24 mg of zearalenone-4O-beta-D-glucopyranoside was obtained from 50 mg of zearalenone and further purified. A total of 10 mg of the glucoside were reduced with sodium borohydride, yielding 4.1 mg alpha-zearalenol-4O-beta-D-glucopyranoside and 4.5 mg beta-zearalenol-4O-beta-D-glucopyranoside at purities higher than 99%. To confirm the identities of the three produced glucosides, MS and MS/MS spectra were acquired using negative electrospray ionization. Besides the deprotonated ions at m/z 479 or 481, respectively, in full-scan mode, fragments, adducts, and dimers were recorded and assigned. MS/MS spectra of the glucosylated substances yielded the deprotonated ions of the mycotoxins zearalenone, alpha-zearalenol, beta-zearalenol and their fragments, respectively. Unambiguous structural assignment of the three substances was achieved using two-dimensional NMR methods. This way, the glucose attachment to position C-4, the beta-configuration of the sugar unit and the stereo-chemical assignment of the zearalenol hydroxyl group at C-6' were proven.

Occurrence of deoxynivalenol and its 3-beta-D-glucoside in wheat and maize.

Deoxynivalenol-3-beta-D-glucoside (D3G), a phase II plant metabolite of the mycotoxin deoxynivalenol (DON), occurs in naturally Fusarium-contaminated cereals. In order to investigate the frequency of occurrence as well as the relative and absolute concentrations of D3G in naturally infected cereals, 23 wheat samples originating from fields in Austria, Germany and Slovakia as well as 54 maize samples from Austrian fields were analysed for DON and D3G by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both analytes were detected in all the 77 field samples. DON was found at levels from 42 to 4130 ng g(-1) (977 +/- 1000 ng g(-1) on average). The D3G concentrations in all cereal samples were in the range 10-1070 ng g(-1) (216 +/- 253 ng g(-1) on average), corresponding to about 5-46 mol% of their DON concentrations (15 +/- 8 mol% on average).

Simultaneous determination of deoxynivalenol, zearalenone, and their major masked metabolites in cereal-based food by LC-MS-MS.

Cereals and cereal-based food have often been found to be contaminated with the mycotoxins deoxynivalenol (DON) and zearalenone (ZON), after infection of the grain with the pathogenic fungus Fusarium. Both the pathogen and the infected plants can chemically modify DON and ZON, including acetylation, glucosidation, and sulfation. Analytical strategies for detection and quantification of DON and ZON are well known and established but often fail to recognize the respective metabolites, which are, therefore, also referred to as "masked" mycotoxins. However, several masked forms are also known to be harmful to mammals. Failure to detect these could lead to significant underestimation of the toxic potential of a particular sample. To monitor the levels of DON and ZON metabolites in cereals and cereal-based food, we have developed a LC-MS-MS method capable of simultaneous determination of DON, ZON, and eight of their masked metabolites, namely deoxynivalenol-3-glucoside (D3G), 3-acetyl-deoxynivalenol (3ADON), zearalenone-4-glucoside (Z4G), alpha-zearalenol (alpha-ZOL), beta-zearalenol (beta-ZOL), alpha-zearalenol-4-glucoside (alpha-ZG), beta-zearalenol-4-glucoside (beta-ZG), and zearalenone-4-sulfate (Z4S). The suitability of several cleanup strategies including C(18)-SPE, primary and secondary amines (PSA), MycoSep push-through columns, and immunoaffinity columns was evaluated. The final method used no sample cleanup and was successfully validated for four cereal-based food matrices, namely cornflour, porridge, beer, and pasta, showing good recoveries and precision for all analytes.