3-acetyldeoxynivalenol and deoxynivalenol-3-glucoside

Topics: Animal Feed; Biotransformation; Chromatography, Liquid; Food Microbiology; Fusarium; Germany; Glucosides; Mass Spectrometry; Risk Assessment; Trichothecenes; Zea mays; Zearalenone; Zeranol

Contamination of wheat grains with Fusarium mycotoxins and their modified forms is an important issue in wheat industry. The objective of this study was to analyse the deoxynivalenol (DON) and deoxynivalenol-3-glucosides (D3G) content in Canadian spring wheat cultivars grown in two locations, inoculated with a mixture of 3-acetyldeoxynivalenol (3-ADON)-producing Fusarium graminearum strains and a mixture of 15-acetlyldeoxynivalenol (15-ADON)-producing F. graminearum strains. According to the analysis of variance, significant differences were observed among the cultivars for Fusarium head blight (FHB) disease index, Fusarium-damaged kernel percentage (%FDK), DON content and D3G content. When the effect of chemotype was considered, significant differences were observed for FHB disease index, FDK percentage and DON content. The D3G content and D3G/DON ratio were not significantly different between the chemotypes, except for D3G content at the Winnipeg location. The Pearson correlation coefficient between DON and D3G was 0.84 and 0.77 at Winnipeg and Carman respectively. The highest D3G/DON ratio was observed in cultivars Carberry (44%) in Carman and CDC Kernen (63.8%) in Winnipeg. The susceptible cultivars showed lower D3G/DON ratio compared with the cultivars rated as moderately resistant and intermediate. The current study indicated that Canadian spring cultivars produce D3G upon Fusarium infection.

Topics: Canada; Disease Resistance; Edible Grain; Food Contamination; Fusarium; Glucosides; Mycotoxins; Plant Diseases; Seasons; Trichothecenes; Triticum

A reliable and sensitive analytical method was developed for simultaneous determination of deoxynivalenol(DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FUS-X), and masked deoxynivalenol (deoxynivalenol-3-glucoside, D3G) in formula feed, concentrated feed, and premixed feed products. The method was based on an improved sample pretreatment with the commercially available HLB cartridges used for sample purification and enrichment followed by analysis using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Several key parameters including the extraction solvents, the positions of sample loading solvents, washing and elution solvents for HLB cartridges were carefully optimized to achieve optimal extraction and purification efficiencies. The established method was extensively validated by determining the linearity (R² ≥ 0.99), sensitivity (limit of quantification in the range of 0.08-4.85 μg/kg), recovery (79.3%-108.1%), precision (Intra-day RSDs ≤ 13.5% and Inter-day RSDs ≤ 14.9%), and then was successfully applied to determine the four type B trichothecenes and D3G in a total of 31 feed samples. Among them, 26 were contaminated with various mycotoxins at the levels of 2.1-864.5 μg/kg, and D3G has also been detected in 17 samples with the concentrations in the range of 2.1-34.8 μg/kg, proving the established method to be a valuable tool for type B trichothecenes and masked DON monitoring in complex feed matrices.

Topics: Chromatography, Liquid; Food Analysis; Glucosides; Humans; Mycotoxins; Tandem Mass Spectrometry; Trichothecenes

The gastrointestinal tract is the first target after ingestion of the mycotoxin deoxynivalenol (DON) via feed and food. Deoxynivalenol is known to affect the proliferation and viability of animal and human intestinal epithelial cells. In addition to DON, feed and food is often co-contaminated with modified forms of DON, such as 3-acetyldeoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) and deoxynivalenol-3-β-D-glucoside (DON3G). The goal of this study was to determine the in vitro intrinsic cytotoxicity of these modified forms towards differentiated and proliferative porcine intestinal epithelial cells by means of flow cytometry. Cell death was assessed by dual staining with Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), which allows the discrimination of viable (FITC-/PI-), apoptotic (FITC+/PI-) and necrotic cells (FITC+/PI+). Based on the data from the presented pilot in vitro study, it is concluded that cytotoxicity for proliferative cells can be ranked as follows: DON3G ≪ 3ADON < DON≈15ADON.

Topics: Animals; Animals, Newborn; Apoptosis; Chromatography, Liquid; Epithelial Cells; Flow Cytometry; Food Contamination; Glucosides; Humans; In Vitro Techniques; Intestines; Magnetic Resonance Spectroscopy; Swine; Tandem Mass Spectrometry; Trichothecenes

A critical assessment of three previously published indirect methods based on acidic hydrolysis using superacids for the determination of "free" and "total" deoxynivalenol (DON) was carried out. The modified mycotoxins DON-3-glucoside (D3G), 3-acetyl-DON (3ADON), and 15-acetyl-DON (15ADON) were chosen as model analytes. The initial experiments focused on the stability/degradation of DON under hydrolytic conditions and the ability to release DON from the modified forms. Acidic conditions that were capable of cleaving D3G, 3ADON, and 15ADON to DON were not found, raising doubts over the efficacy of previously published indirect methods for total DON determination. Validation of these indirect methods for wheat, maize, and barley using UHPLC-MS/MS was performed in order to test the accuracy of the generated results. Validation data for DON, D3G, 3ADON, and 15ADON in nonhydrolyzed and hydrolyzed matrices were obtained. Under the tested conditions, DON was not released from D3G, 3ADON, or 15ADON after hydrolysis and thus none of the published methods were able to cleave the modified forms of DON. In addition to acids, alkaline hydrolysis with KOH for an extended time and at elevated temperatures was also tested. 3ADON and 15ADON were cleaved under the alkaline pH caused by the addition of KOH or aqueous K2CO3 to "neutralize" the acidic sample extracts in the published studies. The published additional DON increase after hydrolysis may have been caused by huge differences in matrix effects and the recovery of DON in nonhydrolyzed and hydrolyzed matrices as well as by the alkaline cleavage of 3ADON or 15ADON after the neutralization of hydrolyzed extracts.

Topics: Chromatography, Liquid; Edible Grain; Glucosides; Hordeum; Hydrolysis; Mycotoxins; Tandem Mass Spectrometry; Trichothecenes; Triticum; Zea mays

In addition to 'traditional' multi-residue and multi-contaminant multiple reaction monitoring (MRM) mass spectrometric techniques devoted to quantifying a list of targeted compounds, the global food industry requires non-targeted methods capable of detecting other possible potentially hazardous compounds. Ultra-high-performance liquid chromatography combined with a single-stage Orbitrap high-resolution mass spectrometer (UHPLC-HRMS Exactive™-Orbitrap Technology) was successfully exploited for the complete selective and quantitative determination of 33 target compounds within three major cross categories (pesticides, antibiotics and mycotoxins) in bakery matrices (specifically milk, wheat flour and mini-cakes). Resolution was set at 50 000 full width at half maximum (FWHM) to achieve the right compromise between an adequate scan speed and selectivity, allowing for the limitations related to the necessary generic sample preparation approach. An exact mass with tolerance of 5 ppm and minimum peak threshold of 10 000 units were fixed as the main identification conditions, including retention time and isotopic pattern as additional criteria devoted to greatly reducing the risk of false-positive findings. The full validation for all the target analytes was performed: linearity, intermediate repeatability and recovery (28 analytes within 70-120%) were positively assessed; furthermore, limits of quantification between 5 and 100 µg kg(-1) (with most of the analytes having a limit of detection below 6 µg kg(-1)) indicate good performance, which is compatible with almost all the regulatory needs. Naturally contaminated and fortified mini-cakes, prepared through combined use of industrial and pilot plant production lines, were analysed at two different concentration levels, obtaining good overall quantitative results and providing preliminary indications of the potential of full-scan HRMS cluster analysis. The effectiveness of this analytical approach was also tested in terms of the formulation of hypotheses for the identification of other analytes not initially targeted which can have toxicological implications (e.g. 3-acetyl-deoxynivalenol and deoxynivalenol-3-glucoside), opening a window on retrospective investigation perspectives in food safety laboratories.

Topics: Animals; Anti-Bacterial Agents; Bread; Chromatography, High Pressure Liquid; Cluster Analysis; Flour; Food Analysis; Food Contamination; Food Safety; Glucosides; Humans; Limit of Detection; Mass Spectrometry; Metabolomics; Milk; Mycotoxins; Pesticides; Phenols; Trichothecenes; Veterinary Drugs

Enzyme-linked immunosorbent assay (ELISA) represents a bioanalytical strategy frequently used for rapid screening of mycotoxin deoxynivalenol (DON) in cereals and derived products. Due to a considerable affinity of some anti-DON antibodies to structurally similar DON metabolites, such as DON-3-glucoside (DON-3-Glc) and 3-acetyl-DON (3-ADON), a significant overestimation of DON concentrations may occur. A validation study of six commercial DON-dedicated ELISA kits, namely Ridascreen DON, Ridascreen FAST, DON, DON EIA, AgraQuant DON Assay, Veratox 5/5, and Veratox HS was carried out on wheat, barley, and malt matrices. Performance characteristics of all tested ELISAs were determined using aqueous solutions of DON, DON-3-Glc, and 3-ADON analytical standards, further with extracts of artificially spiked blank cereals, and finally with matrix-matched standards of all three compounds. In the final phase, the accuracy of data was assessed through a comparison of DON concentrations determined by particular ELISAs and reference ultra-high-performance liquid chromatography-tandem mass spectrometry method. For this purpose, both quality control materials and a comprehensive set of naturally and artificially contaminated samples of wheat, barley, and malt were analyzed. High cross-reactivities were proved for both DON-3-Glc and 3-ADON in the majority of examined assays, and moreover, a considerable contribution of some matrix components to overestimation of DON results was confirmed.

Topics: Antibodies; Antibody Specificity; Artifacts; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Glucosides; Hordeum; Mycotoxins; Reagent Kits, Diagnostic; Tandem Mass Spectrometry; Trichothecenes; Triticum

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.

Topics: DNA, Fungal; Ergosterol; Fusarium; Genotype; Germany; Glucosides; Oryza; Phenotype; Polymerase Chain Reaction; Trichothecenes; Zearalenone