Determination of aflatoxin biomarkers in excreta and ileal content of chickens.

Aflatoxins are carcinogenic secondary metabolites frequently detected in food and feed stuff based on maize and other crops susceptible to infection with the fungal pathogen Aspergillus flavus. We investigated the metabolization of aflatoxins in chickens by analyzing excreta and ileal content and developed and validated a biomarker method for detection of aflatoxins and their metabolites in these matrices. Analysis of ileal content served to distinguish between urinary and fecal excretion combined in the excreta samples. During a 3-wk animal trial, one hundred sixty-eight 1-day-old chicks were randomly allocated to 24 pens with 7 chicks per pen and subjected to different feed regimens with: A) toxin-free feed, B) feed supplemented with 18 ng of total aflatoxins/g, and C) feed supplemented with 515 ng of total aflatoxins/g. Chicken excreta and ileal content were sampled after 7, 14, and 21 D. An analytical method based on liquid chromatography coupled to tandem mass spectrometry was validated for the determination of aflatoxin B1, B2, G1, G2, M1, P1, Q1, and aflatoxin B1-N7-guanine (AFB1-N7-Gua) in chicken's samples. Comparing chicken excreta, which contain urine and feces, to ileal content, which contains no urine, we explored the secretion pathway of aflatoxin metabolites. The AFB1-N7-Gua was only detected in excreta, whereas aflatoxin M1 (AFM1) was detected both in ileal content and excreta. Aflatoxin M1 was detected in excreta in concentrations 5 times higher than in ileal content, suggesting primary excretion via urine. Although chickens are relatively resistant to aflatoxins, contamination of feed can lead to adverse effects and thus economic losses in farming. Therefore, a biomarker method to estimate the exposure of chickens to aflatoxins can play an important role to monitor the animals' health.

Urinary deoxynivalenol (DON) and zearalenone (ZEA) as biomarkers of DON and ZEA exposure of pigs.

Four diets contaminated with 1.1 to 5.0 mg/kg deoxynivalenol (DON) and 0.4 to 2.4 mg/kg zearalenone (ZEA) were fed to four groups of six growing Large White pigs. Urine samples were collected after 3 to 4 days and again after 6 to 7 days on the diets. On each sampling day, half of the animals were sampled in the morning, after an 8-h fast, and the other half were sampled in the afternoon, after 7 h of ad libitum access to feed. The urinary concentrations of DON, DON-glucuronide, DON-3-sulphate, de-epoxy-DON, as well as of ZEA, ZEA-14-glucuronide, α-zearalenol and α-zearalenol-14-glucuronide, analysed using LC-MS/MS, were used to calculate urinary DON and ZEA equivalent concentrations (DONe and ZEAe). The urinary concentration of DONe (P < 0.001), but not of ZEAe (P = 0.31), was lower in the fasted than that in the fed animals. The urinary DONe/creatinine and ZEAe/creatinine ratios were highly correlated with DON and ZEA intake per kg body weight the day preceding sampling (r = 0.76 and 0.77; P < 0.001). The correlations between DON intake during the 7 h preceding urine sampling in the afternoon and urinary DONe/creatinine ratio (r = 0.88) as well as between mean ZEA intake during 3 days preceding urine sampling and urinary ZEAe/creatinine ratio (r = 0.84) were even higher, reflecting the plasma elimination half-time of several hours for DON and of more than 3 days for ZEA. ZEAe analysed in enzymatically hydrolysed urine using an ELISA kit was highly correlated with the LC-MS/MS data (r = 0.94). The urinary DONe and ZEAe to creatinine ratios, analysed in pooled urine samples of several pigs fed the same diet, can be used to estimate their exposure to DON and ZEA.

Prevalence and effects of mycotoxins on poultry health and performance, and recent development in mycotoxin counteracting strategies.

Extensive research over the last couple of decades has made it obvious that mycotoxins are commonly prevalent in majority of feed ingredients. A worldwide mycotoxin survey in 2013 revealed 81% of around 3,000 grain and feed samples analyzed had at least 1 mycotoxin, which was higher than the 10-year average (from 2004 to 2013) of 76% in a total of 25,944 samples. The considerable increase in the number of positive samples in 2013 may be due to the improvements in detection methods and their sensitivity. The recently developed liquid chromatography coupled to (tandem) mass spectrometry allows the inclusion of a high number of analytes and is the most selective, sensitive, and accurate of all the mycotoxin analytical methods. Mycotoxins can affect the animals either individually or additively in the presence of more than 1 mycotoxin, and may affect various organs such as gastrointestinal tract, liver, and immune system, essentially resulting in reduced productivity of the birds and mortality in extreme cases. While the use of mycotoxin binding agents has been a commonly used counteracting strategy, considering the great diversity in the chemical structures of mycotoxins, it is very obvious that there is no single method that can be used to deactivate mycotoxins in feed. Therefore, different strategies have to be combined in order to specifically target individual mycotoxins without impacting the quality of feed. Enzymatic or microbial detoxification, referred to as "biotransformation" or "biodetoxification," utilizes microorganisms or purified enzymes thereof to catabolize the entire mycotoxin or transform or cleave it to less or non-toxic compounds. However, the awareness on the prevalence of mycotoxins, available modern techniques to analyze them, the effects of mycotoxicoses, and the recent developments in the ways to safely eliminate the mycotoxins from the feed are very minimal among the producers. This symposium review paper comprehensively discusses the above mentioned aspects.

Development and validation of an LC-MS/MS method for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin and some masked metabolites in different cereals and cereal-derived food.

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, ß-zearalenol, zearalenone-4-glucoside, α-zearalenol-4-glucoside, ß-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.

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.

Fusarium toxins and total fungal biomass indicators in naturally contaminated wheat samples from north-eastern Poland in 2003.

Concentrations of fungal metabolites were measured in 32 wheat grain samples from north-eastern Poland in 2003. The samples originated from fields cultivated conventionally (but varying in chemical protection level) or cultivated organically. Concentrations of Fusarium toxins (HT-2, DON, 3-AcDON, NIV), trichodiene, microbial biomass indicators (fungal ergosterol and general adenosine 5'-triphosphate (ATP)) and seed vigour were assessed. A large variation between samples was observed, depending on their origin. Seed from organic farms contained similar amounts of Fusarium toxins but more ergosterol and ATP than conventionally grown and chemically protected seed. The highest levels of toxins and ergosterol were detected in samples from conventional cultivation lacking chemical protection. Intensive agronomic practices (including complete chemical protection) significantly lowered the levels of ergosterol, ATP and trichodiene, as compared with other cultivation systems.

Characterization of (13C24) T-2 toxin and its use as an internal standard for the quantification of T-2 toxin in cereals with HPLC-MS/MS.

In this paper, the structure and the identity of fully 13C-substituted T-2 toxin were confirmed using high-resolution mass spectrometry, 1H-NMR, 13C-NMR, tandem mass spectrometry and HPLC-DAD. The purity of this compound was estimated to be at least 98.8% according to UV data. The isotopic distribution of (13C(24)) T-2 toxin indicated a total isotopic enrichment of 98.2 +/- 1.0 atom% 13C, and the application of different MS measurement modes revealed the MS/MS fragmentation pattern of T-2 toxin. Furthermore, a stable isotope dilution mass spectrometry method for the quantification of T-2 toxin was developed using (13C(24)) T-2 toxin as internal standard. The method was evaluated with and without conventional clean-up and validated for maize and oats. Both cereals showed strong matrix enhancement effects, which could be compensated for through the application of the isotope-substituted internal standard.

Short review: Metabolism of theFusarium mycotoxins deoxynivalenol and zearalenone in plants.

Plants have a high capacity to transform and thereby detoxify deleterious or poisonous compounds, like mycotoxins. The formation of glucose conjugates has a central role in this process. Mammals, however, are able to (partly) release the precursor substances during digestion, reactivating the mycotoxins. This short review provides a brief summary about the metabolism of theFusarium mycotoxins deoxynivalenol and zearalenone in plants. Two examples are discussed in greater detail. First, the formation of deoxynivalenol-3-glucoside in wheat is linked to a quantitative trait locus that is often used forFusarium head blight resistance breeding. Secondly, the metabolism of zearalenone inArabidopsis thaliana results in at least 17 different metabolites, all of which are potentially hazardous for humans and animals.

Production of zearalenone-4-glucoside, a-zearalenol-4-glucoside and ß-zearalenol-4-glucoside.

The work at hand describes the production of the zearalenone (ZON) metabolites zearalenone-4-glucoside (ZON-4G), a-zearalenol-4-glucoside (oc-ZOL-4G) and ß-zearalenol-4-glucoside (ß-ZOL-4G). In a first step a genetically modified yeast strain, expressing theArabidopsis thaliana UDP-glu-cosyltransferase UGT73C6, was treated with ZON to produce ZON-4G. The substance was purified by solid phase extraction and subsequent reversed phase preparative HPLC prior to the reduction with sodium borohydride to yield 0C-ZOL-4G and ß-ZOL-4G. The identity and purity of the substances were confirmed by(13)C-and(1)H-NMR as well as by HPLC-UV. In total, 50 mg of ZON were used to produce 5 mg of a-ZOL-4G with a purity of 98%, 6 mg of ß-ZOL-4G with a purity of 99% and 5 mg of ZON-4G with a purity of 99%.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) determination of phase II metabolites of the mycotoxin zearalenone in the model plant Arabidopsis thaliana.

The biotransformation products of zearalenone, a Fusarium mycotoxin, were elucidated using the model plant Arabidopsis thaliana. After treatment of plant seedlings with 50 microM zearalenone, both the liquid media and the plant extracts were analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). An array of 17 different metabolites, most prominently glucosides, malonylglucosides, di-hexose- and hexose-pentose disaccharides of zearalenone, and alpha- and beta-zearalenol, were detected in the samples. Time courses for the different zearalenone metabolites were recorded and they give a closer insight into the metabolism kinetics. A scheme proposing the zearalenone metabolism in A. thaliana is given. The aspect of food safety regarding the (potential) occurrence of masked mycotoxins in agricultural commodities is discussed.

Characterization and application of isotope-substituted (13C15)-deoxynivalenol (DON) as an internal standard for the determination of DON.

The powerful combination of liquid chromatography and mass spectrometry (MS) is often limited by matrix effects during ionization in the MS ion source. The use of fully isotope-substituted (13C15)-deoxynivalenol ((13C15)-DON) as an internal standard (IS) corrects matrix effects and improves the accuracy of analytical methods using mass spectrometry for the quantitative determination of the Fusarium mycotoxin deoxynivalenol (DON). The IS was characterized with respect to its chromatographic purity by liquid chromatography-ultraviolet light and its isotope distribution by time-of-flight mass spectrometry. Its low-energy collision-induced dissociation behaviour was compared with DON. Moreover, this work describes the successful application of (13C15)-DON as IS for the determination of DON in maize using high-performance liquid chromatography (HPLC) electrospray (ESI) with tandem mass spectrometry. The results demonstrate that the IS can successfully correct for fluctuations during extraction and clean-up of the sample as well as the ionization of DON in the MS ion source. Random variations in ionization affect the IS in the same way as the analyte. Recoveries for DON in maize of 76% +/- 1.9% (external calibration) or 101% +/- 2.4% (internal calibration) were reached, respectively, after sample clean-up.

Suitability of a fully 13C isotope labeled internal standard for the determination of the mycotoxin deoxynivalenol by LC-MS/MS without clean up.

Very often, the accuracy of quantitative analytical methods for the determination of mycotoxins by liquid chromatography (LC)-mass spectrometry (MS) and LC-MS/MS is limited by matrix effects during the ionization process in the MS source. Stable isotope labeled standards are best suited to correct for matrix effects and to improve both the trueness and the precision of analytical methods employing LC-MS and LC-MS/MS. This paper describes the successful use of fully 13C isotope labeled deoxynivalenol [(13C15)DON] as an internal standard (IS) for the accurate determination of DON in maize and wheat by LC electrospray ionization MS/MS. To show the full potential of (13C15)DON as IS, maize and wheat extracts were analyzed without further cleanup. Subsequent to calibration for the LC-MS end determination, DON was quantified in matrix reference materials (wheat and maize). Without consideration of the IS, apparent recoveries of DON were 29+/-6% (n=7) for wheat and 37+/-5% (n=7) for maize. However, the determination of DON in the reference materials yielded 95+/-3% (wheat) and 99+/-3% (maize) when (13C15)DON was used as an IS for data evaluation.

Advances in the analysis of mycotoxins and its quality assurance.

This article covers the latest activities in mycotoxin analysis and the advances of its respective quality assurance. The majority of mycotoxin analyses carried out in the laboratories is still based on physicochemical methods, which are continually improved. For example, immunoaffinity columns and multifunctional clean-up columns have become of increasing importance and in some areas of mycotoxin analysis they have more or less displaced conventional liquid-liquid partitioning or column chromatography during clean-up. The need for rapid yes/no decisions on the other hand has led to a number of new screening methods. In particular, rapid and easy-to-use test kits based on immunoanalytical principles or the generation of artificial macromolecular receptors employed in molecularly imprinted polymers (MIPs) have made good progress. Further research in mycotoxin analysis is pursued in the field of biosensors and also the potential of infrared spectroscopic techniques as screening method has been demonstrated. In the area of multi mycotoxin analysis the most promising development was observed in mass spectrometry. At the same time, several interlaboratory studies in the field of mycotoxin analysis revealed problems proven by high between laboratory standard deviation and non-traceable results. This not only shows the necessity of reliable methods and well defined performance characteristics but also the need for appropriate calibrants of defined concentration and stated purity. A certified zearalenone (ZON) calibrant is already available and a certified calibrant containing various trichothecenes is currently under development. (Certified) reference materials are available for aflatoxins in a number of commodities, ochratoxin A (OTA) in wheat, deoxynivalenol (DON) in maize and wheat, and ZON in maize. With these measures important steps towards traceability of results in mycotoxin analysis have been achieved.

Processing and purity assessment of standards for the analysis of type-B trichothecene mycotoxins.

The lack of reliable, certified calibrant solutions for the Fusarium mycotoxins deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), 15-acetyl-DON (15-Ac-DON) and nivalenol (NIV) is a serious drawback in the already problematic area of trichothecene analysis. For this reason, purified DON, 3-Ac-DON, 15-Ac-DON and NIV standards were processed, the conditions required for their isolation and purification were optimised, and the crystalline toxins were thoroughly characterised. Several complimentary analytical methods were used to evaluate the identities of the mycotoxins and the types and amounts of impurities; results obtained from 1H and 13C NMR spectra, as well as from IR-spectra, were in agreement with the literature. Elemental analysis revealed that the isolated NIV occurs as monohydrate. If this is not known it results in a weighing error of approximately 5%. Differential scanning calorimetry (DSC) was only successful for 15-Ac-DON, as the other trichothecenes decomposed during measurements. No traces of chloride, nitrate and sulphate were found by means of ion chromatography (IC). As expected UV absorption spectra for DON, NIV, 3-Ac-DON and 15-Ac-DON yielded lambda(max) values of 216, 217, 217 and 219 nm, respectively. Minor peaks due to impurities were observed by high performance liquid chromatography (HPLC) with UV detection. The main impurity peak in the DON sample was identified by LC-tandem mass spectroscopy (LC-MS/MS) as 4,7-dideoxy-NIV (7-deoxy-DON), which occurs at levels of approximately 1.4%. Gas chromatography (GC) was performed, coupled with either an electron capture detector (ECD), a flame ionisation detector (FID), or a mass spectrometric detector (MS); however, derivatisation prior to GC analysis makes the estimation of impurities difficult. LC-MS/MS was found to be unsuitable for quantifying levels of impurities. It can be concluded that high-purity (>97%) B-trichothecene standards were successfully processed and fully characterised for the first time.

First results of GEN-AU: Cloning of Deoxynivalenol- and Zearalenone-inactivating UDP-glucosyltransferase genes fromArabidopsis thaliana and expression in yeast for production of mycotoxin-glucosides.

First results of the GEN-AU pilot project "Fusarium virulence and plant resistance mechanisms" are reported. Employing genetically engineered yeast strains we have been able to clone genes from the model plantArabidopsis thaliana encoding UDP-glucosyltransferases which can inactivate deoxynivalenol (DON) and zearalenone (ZON). The structure of the metabolites produced by the transformed yeast strains were determined by LC-MS/MS as DON-3O-glucoside and ZON-4O-glucoside, respectively. ZON and derivatives added to glucosyltransferase expressing yeast cultures are converted into the corresponding glucosides in very high yield, opening an efficient way to produce reference materials for these masked mycotoxins.

DON-glycosides: Characterisation of synthesis products and screening for their occurrence in DON-treated wheat samples.

DON-3ß-glucopyranoside was synthesized and characterized by LC-MS/MS. The formation of by-products in the chemical synthesis was further evaluated and, according to their different fragmentation behaviours, it was possible to postulate their structures. The occurrence of these substances in nature was investigated by the analysis of wheat ears, which were treated with DON at anthesis. For the first time DON-3-glucoside could be detected in wheat, strongly indicating that it could also occur in naturally contaminated cereals.

[Determination of DON-3-Glucoside in artificially and naturally contaminated wheat with LC-MS/MS]. / Bestimmung von DON-3-Glukosid in künstlich und natürlich kontaminiertem Weizen mittels LC-MS/MS.

Naturally contaminated and artificiallyFusarium spp. inoculated wheat was analyzed for deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol. After extraction and clean-up with MycoSep columns, the trichothecenes were determined using a LC-ESI-MS/MS method. Deoxynivalenol-3-glucoside was detectable in 4 out of 4 artificially inoculated and in 22 out of 25 naturally contaminated wheat samples. For the latter, the average relative deoxynivalenol-3-glucoside concentration was about 6% of the deoxynivalenol concentration. The maximum relative deoxynivalenol-3-glucoside concentration was 12% as compared to the concentration of deoxynivalenol. In all samples, the concentration of deoxynivalenol-3-glucoside was higher than the concentrations of 3-acetyl-deoxynivalenol or 15-acetyl-deoxynivalenol.