Mycotoxin-mixture assessment in mother-infant pairs in Nigeria: From mothers' meal to infants' urine.

Exposure to food and environmental contaminants is a global environmental health issue. In this study, innovative LC-MS/MS approaches were applied to investigate mycotoxin co-exposure in mother-infant pairs (n = 23) by analyzing matched plate-ready food, breast milk and urine samples of mothers and their exclusively breastfed infants. The study revealed frequent co-occurrence of two to five mycotoxins. Regulated (e.g. aflatoxins, deoxynivalenol and ochratoxin A) and emerging mycotoxins (e.g. alternariol monomethyl ether and beauvericin) were frequently detected (3 %-89 % and 45 %-100 %), in at least one specimen. In addition, a moderate association of ochratoxin A in milk to urine of mothers (r = 0.47; p = 0.003) and infants (r = 0.52; p = 0.019) but no other significant correlations were found. Average concentration levels in food mostly did not exceed European maximum residue limits, and intake estimates demonstrated exposure below tolerable daily intake values. Infants were exposed to significantly lower toxin levels compared to their mothers, indicating the protective effect of breastfeeding. However, the transfer into milk and urine and the resulting chronic low-dose exposure warrant further monitoring. In the future, occurrence of mycotoxin-mixtures, and their combined toxicological effects need to be comprehensively considered and implemented in risk management strategies. These should aim to minimize early-life exposure in critical developmental stages.

Dietary Risk Assessment and Consumer Awareness of Mycotoxins among Household Consumers of Cereals, Nuts and Legumes in North-Central Nigeria.

This study characterized the health risks due to the consumption of mycotoxin-contaminated foods and assessed the consumer awareness level of mycotoxins in households in two north-central Nigerian states during the harvest and storage seasons of 2018. Twenty-six mycotoxins and 121 other microbial and plant metabolites were quantified by LC-MS/MS in 250 samples of cereals, nuts and legumes. Aflatoxins were detected in all food types (cowpea, maize, peanut and sorghum) except in millet. Aflatoxin B1 was the most prevalent mycotoxin in peanut (64%) and rice (57%), while fumonisin B1 occurred most in maize (93%) and beauvericin in sorghum (71%). The total aflatoxin concentration was highest in peanut (max: 8422 µg/kg; mean: 1281 µg/kg) and rice (max: 955 µg/kg; mean: 94 µg/kg), whereas the totals of the B-type fumonisins and citrinin were highest in maize (max: 68,204 µg/kg; mean: 2988 µg/kg) and sorghum (max: 1335 µg/kg; mean: 186 µg/kg), respectively. Citrinin levels also reached 51,195 µg/kg (mean: 2343 µg/kg) in maize. Aflatoxin and citrinin concentrations in maize were significantly (p < 0.05) higher during storage than at harvest. The estimated chronic exposures to aflatoxins, citrinin and fumonisins were high, resulting in as much as 247 new liver cancer cases/year/100,000 population and risks of nephrotoxicity and esophageal cancer, respectively. Children who consumed the foods were the most vulnerable. Mycotoxin co-occurrence was evident, which could increase the health risk of the outcomes. Awareness of mycotoxin issues was generally low among the households.

Human dietary exposure to chemicals in sub-Saharan Africa: safety assessment through a total diet study.

BACKGROUND: Human dietary exposure to chemicals can result in a wide range of adverse health effects. Some substances might cause non-communicable diseases, including cancer and coronary heart diseases, and could be nephrotoxic. Food is the main human exposure route for many chemicals. We aimed to assess human dietary exposure to a wide range of food chemicals. METHODS: We did a total diet study in Benin, Cameroon, Mali, and Nigeria. We assessed 4020 representative samples of foods, prepared as consumed, which covered more than 90% of the diet of 7291 households from eight study centres. By combining representative dietary surveys of countries with findings for concentrations of 872 chemicals in foods, we characterised human dietary exposure. FINDINGS: Exposure to lead could result in increases in adult blood pressure up to 2·0 mm Hg, whereas children might lose 8·8-13·3 IQ points (95th percentile in Kano, Nigeria). Morbidity factors caused by coexposure to aflatoxin B1 and hepatitis B virus, and sterigmatocystin and fumonisins, suggest several thousands of additional liver cancer cases per year, and a substantial contribution to the burden of chronic malnutrition in childhood. Exposure to 13 polycyclic aromatic hydrocarbons from consumption of smoked fish and edible oils exceeded levels associated with possible carcinogenicity and genotoxicity health concerns in all study centres. Exposure to aluminium, ochratoxin A, and citrinin indicated a public health concern about nephropathies. From 470 pesticides tested across the four countries, only high concentrations of chlorpyrifos in smoked fish (unauthorised practice identified in Mali) could pose a human health risk. INTERPRETATION: Risks characterised by this total diet study underscore specific priorities in terms of food safety management in sub-Saharan Africa. Similar investigations specifically targeting children are crucially needed. FUNDING: Standards and Trade Development Facility.

Towards a dietary-exposome assessment of chemicals in food: An update on the chronic health risks for the European consumer.

An informed opinion to a hugely important question, whether the food on the Europeans' plate is safe to eat, is provided. Today, the Europeans face food-borne health risks from non-communicable diseases induced by excess body weight, outbreaks caused by pathogens, antimicrobial resistance and exposures to chemical contaminants. In this review, these risks are first put in an order of importance. Then, not only potentially injurious dietary chemicals are discussed but also beneficial factors of the food. This review can be regarded as an attempt towards a dietary-exposome evaluation of the chemicals, the average European adult consumers could chronically expose to during their life-times. Risk ranking reveals that currently the European adults are chronically exposed to a mixture of potentially genotoxic-carcinogenic contaminants, particularly food process contaminants, at the potential risk levels. Furthermore, several of the contaminants whose dietary exposures pose risks appear to be carcinogens operating with a genotoxic mode of action targeting the liver. This suggests that combined health risks from the exposure to a mixture of the chemical contaminants poses a greater potential risk than the risks assessed for single compounds. Over 100 European-level risk assessments are examined. Finally, the importance of a diversified and balanced diet is emphasized.

Mycotoxin and cyanogenic glycoside assessment of the traditional leafy vegetables mutete and omboga from Namibia.

Sixty traditional leafy vegetables, comprising of mutete (Hibiscus sabdariffa) (n = 20) and omboga (Cleome gynandra) (n = 40) were analysed for fungal, plant and bacterial metabolites using liquid-chromatography-tandem mass spectrometry. No European Union legislated mycotoxins were quantified and no vegetables contained levels above the FAO/WHO limit of 10 mg/kg for cyanogenic potential, suggesting comparative safety regarding regulated mycotoxins and cyanogenic glycosides. Quantified fungal metabolites included averufin and 3-Nitropropionic acid from Aspergillus flavus, beauvericin and equisetin from Fusarium, citrinin and curvularin from Penicillium and altertoxin -1 and tentoxin from Alternaria. Of the plant cyanogenic glycosides, linamarin was quantifiable in 65% of mutete at a maximum of 398 µg/kg but not in omboga, while lotaustralin was quantifiable in both omboga and mutete. The bacterial metabolite nonactin was detected in 27.5% of omboga samples (range: 0.2-7.3 µg/kg). Minimal variation in metabolite patterns was recorded for omboga samples from Oshana and Oshikoto regions.

Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis.

Molecular diagnostic tools in the field of food and water quality analysis are becoming increasingly widespread. Usually, based on DNA amplification techniques such as polymerase chain reaction (PCR), these methods are highly sensitive and versatile but require well-equipped laboratories and trained personnel. To reduce analysis time and avoid expensive equipment, isothermal DNA amplification methods for detecting various target organisms have been developed. However, to make molecular diagnostics suitable for low-resource settings and in-field applications, it is crucial to continuously adapt the working steps associated with DNA amplification, namely sample preparation, DNA extraction, and visualization of the results. Many novel approaches have been evaluated in recent years to tackle these challenges, e.g., the use of ionic liquids for the rapid isolation of nucleic acids from organisms relevant for food and water analysis or the integration of entire analytical workflows on microfluidic chips. In any event, the future of applications in the field of isothermal amplification will probably lie in ready-to-use cartridges combined with affordable handheld devices for on-site analysis. This trend article aims to make prospective users more familiar with this technology and its potential for moving molecular diagnostics from the laboratory to the field. Graphical abstract ᅟ.

From malt to wheat beer: A comprehensive multi-toxin screening, transfer assessment and its influence on basic fermentation parameters.

The aim was to determine the mycotoxin transfer rate into beer during a semi-industrial production process and the effect of fungicide treatment in the field on mycotoxins concentrations in beer. To ensure the usual practical agronomical conditions, sample A was treated with fungicide Prosaro® 250, and sample B was infected with Fusarium culmorum spores, in order to obtain infected malt. Malt was produced using standard procedure and beer was produced in a semi-industrial unit. During fermentation measurement of sugars (maltotriose and maltose), glycerol and ethanol content was performed on a daily basis. Multiple toxins were determined in malt and beer. Deoxynivalenol (DON), its modified plant metabolite DON-3-glucoside (DON-glucoside), brevianamide F, tryptophol, linamarin, lotaustralin, culmorin (CUL), 15-hydroxy-CUL and 5-hydroyx-CUL were detected in all samples. Results indicate that F. culmorum infection did not influence the fermentation process or the alcohol concentration.

Traditionally Processed Beverages in Africa: A Review of the Mycotoxin Occurrence Patterns and Exposure Assessment.

African traditional beverages are widely consumed food-grade liquids processed from single or mixed grains (mostly cereals) by simple food processing techniques, of which fermentation tops the list. These beverages are very diverse in composition and nutritional value and are specific to different cultures and countries. The grains from which home-processed traditional beverages are made across Africa are often heavily contaminated with multiple mycotoxins due to poor agricultural, handling, and storage practices that characterize the region. In the literature, there are many reports on the spectrum and quantities of mycotoxins in crops utilized in traditional beverage processing, however, few studies have analyzed mycotoxins in the beverages themselves. The available reports on mycotoxins in African traditional beverages are mainly centered on the finished products with little information on the process chain (raw material to final product), fate of the different mycotoxins during processing, and exposure estimates for consumers. Regulations targeting these local beverages are not in place despite the heavy occurrence of mycotoxins in their raw materials and the high consumption levels of the products in many homes. This paper therefore comprehensively discusses for the 1st time the available data on the wide variety of African traditional beverages, the mycotoxins that contaminate the beverages and their raw materials, exposure estimates, and possible consequent effects. Mycotoxin control options and future directions for mycotoxin research in beverage production are also highlighted.

Mycotoxin risk assessment for consumers of groundnut in domestic markets in Nigeria.

The fungal and multi-mycotoxin profiles of groundnuts sold in domestic markets in Nigeria as well as the associated risk to consumers were assessed in the present study. Four hundred fungal isolates representing mainly Aspergillus [58.6%: Aspergillus section Flavi (37.1%) and A. niger-clade (21.5%)], Penicillium (40.9%) and Fusarium (0.5%) were isolated from 82 (97.6%, n=84) groundnut samples collected from four agro-ecological zones (AEZs) of Nigeria. The incidence of aflatoxin-producing A. flavus isolates (71%) was significantly (p<0.05) higher in the groundnuts than that of the non-aflatoxigenic isolates (29%). Fifty-four fungal metabolites [including aflatoxins (AFB1, AFB2, AFG1, AFG2 and AFM1), beauvericin (BEAU), cyclopiazonic acid (CPA), moniliformin, nivalenol and ochratoxin A] and four bacterial metabolites were detected in the groundnuts by liquid chromatography tandem mass spectrometry. Aflatoxins (39%; max: 2076µg/kg; mean: 216µg/kg) were detected in more samples than any other mycotoxin. About 25, 23 and 14% of the samples respectively were above the 2µg/kg AFB1, 4 and 20µg/kg total aflatoxin limits of the European Union and US FDA respectively. The mean margins of exposure of AFB1 and total aflatoxins for adult consumers were 1665 and 908, respectively, while mean estimated daily intake values for infants, children and adults were <0.1% for BEAU and 4% for CPA. Consumers of mycotoxin contaminated groundnuts in Nigeria may therefore be at a risk of liver cancer in addition to other combinatory effects of mycotoxin/metabolite cocktails. There is need for increased targeted interventions in the groundnut value chain in Nigeria for public health benefits.

Mycotoxin Contamination in Sugarcane Grass and Juice: First Report on Detection of Multiple Mycotoxins and Exposure Assessment for Aflatoxins B1 and G1 in Humans.

This study was conducted to investigate the natural co-occurrence of multiple toxic fungal and bacterial metabolites in sugarcane grass and juice intended for human consumption in Upper Egypt. Quantification of the target analytes has been done using the "dilute and shoot" approach followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total number of 29 and 33 different metabolites were detected in 21 sugarcane grass and 40 juice samples, respectively, with a trend of concentrations being higher in grass than in juice. Among the regulated mycotoxins, only aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) were detected. The prevalence of AFB1 was in 48% of grass samples and in 58% of juice with a maximum concentration of 30.6 µg/kg and 2.10 µg/kg, respectively. AFG1 was detected in 10% of grass samples (7.76 µg/kg) and 18% of juice samples (34 µg/kg). Dietary exposure was assessed using a juice frequency questionnaire of adult inhabitants in Assiut City. The assessment revealed different levels of exposure to AFB1 between males and females in winter and summer seasons. The estimated seasonal exposure ranged from 0.20 to 0.40 ng/kg b.w./day in winter and from 0.38 to 0.90 ng/kg b.w./day in summer.

Mould and mycotoxin exposure assessment of melon and bush mango seeds, two common soup thickeners consumed in Nigeria.

An examination of the mould and fungal metabolite pattern in melon and bush mango seeds locally produced in Nigeria was undertaken in order to understand the mycotoxicological risk posed to consumers of both of these important and commonly consumed soup thickeners. The variation in mycotoxin levels in graded categories of both foodstuffs were also determined. Aspergillus, Fusarium, Penicillium, Mucorales and Trichoderma were the recovered fungi from the foodstuffs with Aspergillus species dominating (melon=97.8%; bush mango=89.9%). Among the Aspergillus species identified Aspergillus section Flavi dominated (melon: 72%; bush mango: 57%) and A. flavus, A. parasiticus, A. parvisclerotigenus and A. tamarii were the recovered species. About 56% and 73% of the A. flavus isolates from melon and bush mango seed samples, respectively were aflatoxigenic. Thirty-four and 59 metabolites including notable mycotoxins were found in the melon and bush mango seeds respectively. Mean aflatoxin levels (µg/kg) in melon (aflatoxin B1 (AFB1)=37.5 and total aflatoxins=142) and bush mango seeds (AFB1=68.1 and total aflatoxins=61.7) were higher than other mycotoxins, suggesting potential higher exposure for consumer populations. Significantly (p<0.05) higher levels of mycotoxins were found in hand-peeled melon and discoloured bush mango seeds than in machine-peeled melon and non-discoloured seeds except for HT-2 and T-2 toxins which occurred conversely. All melon and bush mango seeds exceeded the 2µg/kg AFB1 limit whereas all melon and 55% of bush mango seeds exceeded the 4µg/kg total aflatoxin EU limit adopted in Nigeria. This is the first report of (1) mycotoxin co-occurrence in bush mango seeds, (2) cyclopiazonic acid, HT-2 toxin, moniliformin, mycophenolic acid, T-2 toxin and tenuazonic acid occurrence, and (3) mycotoxin exposure assessment of both foodstuffs.

Penicillium strains isolated from Slovak grape berries taxonomy assessment by secondary metabolite profile.

The secondary metabolite profiles of microfungi of the genus Penicillium isolated from samples of grape berries collected in two different phases during two vegetative seasons in Slovakia is described to assess the taxonomy. Three Slovak vine regions have been selected for this study, based on their climatic differences and national economic importance. Cultures of microfungi isolated from berries were incubated on different selective media for macro and micromorphology identification. The species Penicillium brevicompactum, Penicillium crustosum, Penicillium chrysogenum, Penicillium expansum, Penicillium palitans and Penicillium polonicum were identified according to growth and morphology. The related strains were found to produce a broad spectrum of fungal metabolites, including roquefortine C, chaetoglobosin A, penitrem A, cyclopeptin, cyclopenin, viridicatin, methylviridicatin, verrucofortine, secalonic acid D, cyclopiazonic acid, fumigaclavine and mycophenolic acid. Chemotaxonomy was performed using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Dried grape berries were also analyzed allowing to assess the presence of patulin, roquefortine C and penicillic acid; this last one has been identified in dried berries but not in vitro.

LC-MS/MS-based multibiomarker approaches for the assessment of human exposure to mycotoxins.

Mycotoxins are toxic fungal secondary metabolites that frequently contaminate food and feed worldwide, and hence represent a major hazard for food and feed safety. To estimate human exposure arising from contaminated food, so-called biomarker approaches have been developed as a complementary biomonitoring tool besides traditional food analysis. The first methods based on radioimmunoassays and enzyme-linked immunosorbent assays as well as on liquid chromatography were developed in the late 1980s and early 1990s for the carcinogenic aflatoxins and in the last two decades further tailor-made methods for some major mycotoxins have been published. Since 2010, there has been a clear trend towards the development and application of multianalyte methods based on liquid chromatography-electrospray ionization tandem mass spectrometry for assessment of mycotoxin exposure made possible by the increased sensitivity and selectivity of modern mass spectrometry instrumentation and sophisticated sample cleanup approaches. With use of these advanced methods, traces of mycotoxins and relevant breakdown and conjugation products can be quantified simultaneously in human urine as so-called biomarkers and can be used to precisely describe the real exposure, toxicokinetics, and bioavailability of the toxins present. In this article, a short overview and comparison of published multibiomarker methods focusing on the determination of mycotoxins and relevant excretion products in human urine is presented. Special attention is paid to the main challenges when analyzing these toxic food contaminants in urine, i.e., very low analyte concentrations, appropriate sample preparation, matrix effects, and a lack of authentic, NMR-confirmed calibrants and reference materials. Finally, the progress in human exposure assessment studies facilitated by these analytical methods is described and an outlook on probable developments and possibilities is presented.

Assessment of human deoxynivalenol exposure using an LC-MS/MS based biomarker method.

The Fusarium toxin deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and poses many adverse health effects to human and animals. Consequently, regulatory limits and a provisional maximum tolerable daily intake (PMTDI) for this important type B-trichothecene were assigned. We conducted a pilot survey to investigate the level of DON exposure in Austrian adults by measurements of DON and its glucuronide conjugates (DON-GlcA's), as biomarkers of exposure, in first morning urine. The average concentration of total DON (free DON+DON-GlcA's) was estimated to be 20.4±2.4 µg L⁻¹ (max. 63 µg L⁻¹). Surprisingly, we found that one third of the volunteers (n=27) exceeded the established PMTDI when consuming regular diet. DON-GlcA's were directly quantified by LC-MS/MS and the results were compared with indirect quantification after enzymatic hydrolysis and confirmed the suitability of the direct method. Moreover, we investigated the in vivo metabolism of DON in humans and were able to determine two closely eluting DON-GlcA's in naturally contaminated urine samples for the first time. In contrast to previous findings we have tentatively identified DON-15-glucuronide as a major DON metabolite in human urine based on the analysis of these samples. About 75% of total glucuronides were derived from this metabolite while DON-3-glucuronide accounted for approximately 25%. The reported new findings clearly demonstrate the great potential of suitable biomarkers to critically assess exposure of humans and animals to DON.

A rapid fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins in wheat.

A rapid fluorescence polarization (FP) immunoassay has been developed for the simultaneous determination of T-2 and HT-2 toxins in naturally contaminated wheat samples. Syntheses of four fluorescein-labelled T-2 or HT-2 toxin tracers were carried out and their binding response with seven monoclonal antibodies was evaluated. The most sensitive antibody-tracer combination was obtained by using an HT-2-specific antibody and a fluorescein-HT-2 tracer. The developed competitive FP immunoassay in solution showed high cross-reactivity for T-2 toxin (CR% = 100%) while a very low CR% for neosolaniol (0.12%) and no cross-reactivity with other mycotoxins frequently occurring in wheat. A rapid extraction procedure using 90% methanol was applied to wheat samples prior to FP immunoassay. The average recovery from spiked wheat samples (50 to 200 µg kg(-1)) was 96% with relative standard deviation generally lower than 8%. A limit of detection of 8 µg kg(-1) for the combined toxins was determined. Comparative analyses of 45 naturally contaminated and spiked wheat samples by both the FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up showed a good correlation (r = 0.964). These results, combined with the rapidity (10 min) and simplicity of the assay, show that this method is suitable for high throughput screening as well as for quantitative determination of T-2 and HT-2 toxins in wheat.

Direct quantification of deoxynivalenol glucuronide in human urine as biomarker of exposure to the Fusarium mycotoxin deoxynivalenol.

The direct quantification of deoxynivalenol glucuronide (DON-GlcA) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and its application as a biomarker of exposure to the Fusarium mycotoxin deoxynivalenol (DON) is reported. Usually, DON exposure is estimated from dietary average intakes or by measurement of the native toxin in urine after enzymatic hydrolysis with ß-glucuronidase. These methods are time-consuming, expensive, and fail to determine the ratio of DON to DON-GlcA in a simple one-step procedure. One of the main reasons for the use of indirect methods is the unavailability of DON-GlcA standards. Consequently, DON-3-O-glucuronide (D3GlcA) was synthesized and used to develop a method allowing quantification of both DON and D3GlcA by a simple "dilute and shoot" approach without the need for any cleanup. Limit of detection and apparent recovery of D3GlcA was 3 µg l(-1) and 88%, respectively. The identity of D3GlcA in human urine was confirmed by comparison with LC-MS/MS measurements of the synthetically produced D3GlcA standard which was also used for external calibration. The applicability of the method was demonstrated through the analysis of urine samples obtained from a volunteer during regular and cereal-restricted diet, respectively. In regular-diet urine samples, D3GlcA was quantified in concentrations >30 µg l(-1) by this approach.

Determination of ergot alkaloids: purity and stability assessment of standards and optimization of extraction conditions for cereal samples.

Results obtained from a purity study on standards of the 6 major ergot alkaloids ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, and ergocornine and their corresponding epimers are discussed. The 6 ergot alkaloids studied have been defined by the European Food Safety Authority as those that are the most common and physiologically active. The purity of the standards was investigated by means of liquid chromatography with diode array detection, electrospray ionization, and time-of-flight mass spectrometry (LC-DAD-ESI-TOF-MS). All of the standards assessed showed purity levels considerably above 98% apart from ergocristinine (94%), ergosine (96%), and ergosinine (95%). Also discussed is the optimization of extraction conditions presented in a recently published method for the quantitation of ergot alkaloids in food samples using solid-phase extraction with primary secondary amine (PSA) before LC/MS/MS. Based on the results obtained from these optimization studies, a mixture of acetonitrile with ammonium carbonate buffer was used as extraction solvent, as recoveries for all analyzed ergot alkaloids were significantly higher than those with the other solvents. Different sample-solvent ratios and extraction times showed just minor influences in extraction efficacy. Finally, the stability of the ergot alkaloids in both raw cereals and cereal-based processed food extracts was studied. According to these studies, extracts should be prepared and analyzed the same day or stored below ambient temperatures. Barley and rye extracts, which were stored at 4 and 15 degrees C after PSA cleanup, proved to be stable overnight. However, storage over a period of 14 days at 4 degrees C resulted in significant epimerization, which was most pronounced in rye and particularly for ergocornine, ergocryptine, and ergocristine.

Optimisation of a sample preparation procedure for the screening of fungal infection and assessment of deoxynivalenol content in maize using mid-infrared attenuated total reflection spectroscopy.

A sample preparation procedure for the determination of deoxynivalenol (DON) using attenuated total reflection mid-infrared spectroscopy is presented. Repeatable spectra were obtained from samples featuring a narrow particle size distribution. Samples were ground with a centrifugal mill and analysed with an analytical sieve shaker. Particle sizes of <100, 100-250, 250-500, 500-710 and 710-1000 microm were obtained. Repeatability, classification and quantification abilities for DON were compared with non-sieved samples. The 100-250 microm fraction showed the best repeatability. The relative standard deviation of spectral measurements improved from 20 to 4.4% and 100% of sieved samples were correctly classified compared with 79% of non-sieved samples. The DON level in analysed fractions was a good estimate of overall toxin content.

Purity assessment of commercially available crystalline deoxynivalenol.

Deoxynivalenol (DON) obtained from 2 commercial sources was characterized, and its purity was determined. The structural identity of DON was confirmed by 1H and 13C-nuclear magnetic resonance (NMR) spectroscopy, gas chromatography with mass spectrometric (GC/MS) detection, and infrared/attenuated total reflectance (IR/ATR) spectroscopy. NMR spectra showed shifts that varied from previously published data. However, we established a complete, unambiguous assignment for all signals. Chromatograms obtained by GC/MS were almost identical for both investigated samples and confirmed the structure of DON. Likewise, IR/ATR spectra verified the identity of DON. The degree of purity was determined by liquid chromatography (LC) with a variable wavelength detector, LC/MS/MS, GC with electron-capture detection (GC-ECD), and ultraviolet (UV) spectrophotometry. The purity check using LC showed a single peak in both chromatograms. With LC/MS/MS measurements, we could detect small amounts of impurities in the crystalline DON from both sources. In data obtained by GC-ECD, no differences in purity were observed. The UV measurements showed an absorption maximum at 217 nm. The mean epsilon(m) of the extinction coefficients was calculated as 6727 (L/cm/mol) for DON (Sigma) and 6825 (L/cm/mol) for DON (Biopure). Finally, the purity of DON from the 2 commercial sources was calculated as >96 and >98%, respectively. Although the DON produced by both providers can be considered sufficiently pure for routine analysis of trichothecenes in food and feed, this work again demonstrated that the impurity of the solid mycotoxin constitutes the greatest contribution to the overall uncertainty of a mycotoxin calibrant.

Purity assessment of crystalline zearalenone.

Commercially available solid zearalenone (ZON) to be used as a certified liquid calibrant (BCR-699) in a project funded by the European Commission within the Standard Measurement and Testing program was characterized and its purity determined. The degree of purity of the ZON was examined by UV spectrophotometer, liquid chromatography (LC) with diode array and fluorescence detection, 1H and 13C-NMR spectrometry, LC-mass spectrometry (LC/MS/MS), ion chromatography (IC), and differential scanning calorimetry (DSC). The diagrams obtained from DSC analysis and the UV spectrum showed no detectable impurities. Likewise, no impurities were observed by LC analysis with both diode array and fluorescence detection. IC determination revealed negligible contamination of ZON with chloride of 0.020 +/- 0.005% and nitrate of 0.016 +/- 0.006%. Zearalanone (ZAN) was identified as one of 2 minor (0.2%) impurities by LC/MS/MS. The 1H-NMR measurements revealed an additional peak, which has not been previously reported in the literature. It could be identified as part of the ZON spectrum as the signal arising from the phenolic proton attached to C4'. The manufacturer states an additional contamination with 0.2% methylene chloride, which could be confirmed to an extent of 0.1% by 1H-NMR. Minor impurities, whose structures remain unknown, were discovered at 3.5 and < 1 ppm. Total percentage of impurities based on NMR measurement was estimated not to exceed 1%. A purity of 99.5% with a tolerance of +/- 0.5% was finally attributed to the ZON studied in this project.