Fusarium mycotoxin content of UK organic and conventional wheat

Each year (2001–2005), 300 samples of wheat from fields of known agronomy were analysed for ten trichothecenes by gas chromatography-mass spectrometry (GC/MS) including deoxynivalenol (DON), nivalenol, 3-acetyl-DON, 15-acetyl-DON, fusarenone X, T2 toxin, HT2 toxin, diacetoxyscirpenol, neosolaniol and T-2 triol and zearalenone by high-performance liquid chromatography (HPLC). Of the eleven mycotoxins analysed from 1624 harvest samples of wheat, only eight were detected, and of these only five–deoxynivalenol, 15-acetyl-DON, nivalenol, HT-2 and zearalenone–were detected above 100 µg kg^?1. DON was the most frequently detected Fusarium mycotoxin, present above the limit of quantification (10 µg kg^?1) in 86% of samples, and was usually present at the highest concentration. The percentage of samples that would have exceeded the recently introduced legal limits varied between 0.4% and 11.3% over the five-year period. There was a good correlation between DON and zearalenone concentrations, although the relative concentration of DON and zearalenone fluctuated between years. Year and region had a significant effect on all mycotoxins analysed. There was no significant difference in the DON concentration of organic and conventional samples. There was also no significant difference in the concentration of zearalenone between organic and conventional samples, however organic samples did have a significantly lower concentration of HT2 and T2. Overall, the risk of UK wheat exceeding the newly introduced legal limits for Fusarium mycotoxins in cereals intended for human consumption is low, but the percentage of samples above these limits will fluctuate between years.     

Transfer of Fusarium mycotoxins and ‘masked’ deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer

The fate of five Fusarium toxins — deoxynivalenol (DON), sum of 15- and 3-acetyl-deoxynivalenol (ADONs), HT-2 toxin (HT-2) representing the main trichothecenes and zearalenone (ZON) during the malting and brewing processes — was investigated. In addition to these ‘free’ mycotoxins, the occurrence of deoxynivalenol-3-glucoside (DON-3-Glc) was monitored for the first time in a beer production chain (currently, only DON and ZON are regulated). Two batches of barley, naturally infected and artificially inoculated with Fusarium spp. during the time of flowering, were used as a raw material for processing experiments. A highly sensitive procedure employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was validated for the analysis of ‘free’ Fusarium mycotoxins and DON-conjugate in all types of matrices. The method was also able to detect nivalenol (NIV), fusarenon-X (FUS-X) and T-2 toxin (T-2); nevertheless, none of these toxins was found in any of the samples. While steeping of barley grains (the first step in the malting process) apparently reduced Fusarium mycotoxin levels to below their quantification limits (5–10 µg kg^?1), their successive accumulation occurred during germination. In malt, the content of monitored mycotoxins was higher compared with the original barley. The most significant increase was found for DON-3-Glc. During the brewing process, significant further increases in levels occurred. Concentrations of this ‘masked’ DON in final beers exceeded ‘free’ DON, while in malt grists this trichothecene was the most abundant, with the DON/DON-3-Glc ratio being approximately 5:1 in both sample series. When calculating mass balance, no significant changes were observed during brewing for ADONs. The content of DON and ZON slightly decreased by a maximum of 30%. Only traces of HT-2 were detected in some processing intermediates (wort after trub removal and green beer).     

Fusarium mycotoxin content of UK organic and conventional barley

Each year (2002–2005), approximately 100 samples of barley from fields of known agronomy were analysed for ten trichothecenes by gas chromatography-mass spectrometry (GC/MS) including deoxynivalenol (DON), nivalenol, 3-acetyl DON, 15-acetyl DON, fusarenone X, T-2 toxin (T2), HT-2 toxin (HT2), diacetoxyscirpenol, neosolaniol, and T-2 triol. Samples were also analysed for moniliformin and zearalenone by high-performance liquid chromatography (HPLC). Of the ten trichothecenes analysed from 446 harvest samples of barley, only two, diacetoxyscirpenol and neosolaniol, were not detected. The concentrations of type A trichothecenes were similar to those that occurred in wheat over the same period, whilst those of type B trichothecenes were markedly lower. Deoxynivalenol was the most frequently detected Fusarium mycotoxin, present above the limit of quantification (10 µg kg^?1) in 57% of samples, and was usually present at the highest concentration. A single sample (0.2%) exceeded the legal limit for DON in unprocessed barley over the 4-year period. Moniliformin and zearalenone were both rarely detected (2% of samples greater than 10 µg kg^?1 for both toxins) with maximum concentrations of 45 and 44 µg kg^?1, respectively. Year and region had a significant effect on DON and HT2 + T2, but there was no significant difference in the concentration of these mycotoxins between organic and conventional samples. Overall, the risk of UK barley exceeding the newly introduced legal limits for Fusarium mycotoxins in cereals intended for human consumption is very low, but the percentage of samples above these limits will fluctuate between years.     

Survey of 11 mycotoxins in wheat flour in Hebei province,China

A survey of 11 mycotoxins in 348 wheat flour samples marketed in Hebei province of China were analysed by liquid chromatography-tandem mass spectrometry, was carried out. The selected mycotoxins consisted of four aflatoxins (AFs: AFB₁, AFB₂, AFG₁ and AFG₂) and seven Fusarium toxins, i.e. deoxynivalenol (DON), nivalenol, 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol, zearalenone, Fusarenon-X and deoxynivalenol-3-glucoside. Results indicated that most of the wheat samples analysed were contaminated with mycotoxins. Wheat was most susceptible to DON (91.4% contamination), with a mean level of 240 μg kg^?1. On average the probable daily intake (PDI, expressed as µg kg^?1 body weight day^?1) of mycotoxins was within the provisional maximum tolerable daily intake (PMTDI, 2.0 µg kg^?1 of body weight day^?1) as set by the Joint FAO/WHO Expert Committee on Food Additives. Nevertheless, exposure assessment revealed that the maximum PDI of mycotoxins was 4.06 µg kg^?1 body weight day^?1, which was twice the PMTDI value. Thus, consistent monitoring is recommended, as to keep the contamination level under control.     

Mycotoxins in wheat flour and intake assessment in Shandong province of China

In the present study, the occurrence and contamination levels of eight mycotoxins were investigated in wheat flour samples (n = 359) from Shandong Province of China. Samples were determined using a multi-mycotoxin method based on isotope dilution ultrahigh performance liquid chromatography–tandem mass spectrometry. The results indicated that the most frequently found mycotoxins were deoxynivalenol (DON) (97.2%), nivalenol (40.4%) and deoxynivalenol-3-glucoside (33.4%), and mean contamination levels in positive samples were 86.7, 3.55 and 3.34 µg kg^?1, respectively. The obtained data were further used to estimate the daily intake of the local population, and indicated that wheat flour consumption contributes little to DON exposure. However, with the aim to keep the contamination levels under control and to establish a more precise evaluation of the mycotoxin burden in Shandong Province, more sample data from different harvest years and seasons are needed in the future.     

Transfer of Fusarium mycotoxins and 'masked' deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer

The fate of five Fusarium toxins--deoxynivalenol (DON), sum of 15- and 3-acetyl-deoxynivalenol (ADONs), HT-2 toxin (HT-2) representing the main trichothecenes and zearalenone (ZON) during the malting and brewing processes--was investigated. In addition to these 'free' mycotoxins, the occurrence of deoxynivalenol-3-glucoside (DON-3-Glc) was monitored for the first time in a beer production chain (currently, only DON and ZON are regulated). Two batches of barley, naturally infected and artificially inoculated with Fusarium spp. during the time of flowering, were used as a raw material for processing experiments. A highly sensitive procedure employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was validated for the analysis of 'free' Fusarium mycotoxins and DON-conjugate in all types of matrices. The method was also able to detect nivalenol (NIV), fusarenon-X (FUS-X) and T-2 toxin (T-2); nevertheless, none of these toxins was found in any of the samples. While steeping of barley grains (the first step in the malting process) apparently reduced Fusarium mycotoxin levels to below their quantification limits (5-10 microg kg(-1)), their successive accumulation occurred during germination. In malt, the content of monitored mycotoxins was higher compared with the original barley. The most significant increase was found for DON-3-Glc. During the brewing process, significant further increases in levels occurred. Concentrations of this 'masked' DON in final beers exceeded 'free' DON, while in malt grists this trichothecene was the most abundant, with the DON/DON-3-Glc ratio being approximately 5:1 in both sample series. When calculating mass balance, no significant changes were observed during brewing for ADONs. The content of DON and ZON slightly decreased by a maximum of 30%. Only traces of HT-2 were detected in some processing intermediates (wort after trub removal and green beer).     

Validation and application of a liquid chromatography-tandem mass spectrometry based method for the assessment of the co-occurrence of mycotoxins in maize silages from dairy farms in NW Spain

The first objective of this study was the validation of an efficient multi-analyte method for the simultaneous detection and quantification of mycotoxins in maize silage, by reverse-phase liquid chromatography coupled with electrospray ionisation triple quadrupole mass spectrometry (LC-HESI-MS/MS). A simple liquid/solid extraction was performed either with clean-up on Mycospin 400 columns or without any clean-up. Almost all the target mycotoxins showed highly-suppressed signals in the presence of a matrix, emphasising the need to quantitate mycotoxins by means of matrix-matched calibrations. An alternative validation method based on ISO 11843 and on a single factor balanced design was implemented. The achieved average recoveries from spiked samples at three levels ranged from 60% to 122% with relative standard deviations (rsd) below 11%. Limits of Detection (LODs) and Limits of Quantification (LOQs) were between 0.02–17.1 µg kg^?1 and 0.06–57 µg kg^?1. The calculated repeatability and within-lab reproducibility ranged from 5.2 to 23.2% and from 7.2 to 23.9%, respectively. Finally, the decision limit and detection capacity, CCα and CCβ, were calculated for all mycotoxins having regulated/recommended contents in feed. The validated method was applied to 148 samples collected over two years in 19 dairy farms from Galicia (NW Spain). Of the analysed samples, 62% contained at least one mycotoxin. Zearalenone (ZEA), deoxynivalenol (DON), fumonisins B1 and B2, roquefortine C, α-zearalenol, β-zearalenol, enniatins B and B1, andrastin A, marcfortine A, verruculogen and mycophenolic acid were quantified, the highest average detection frequency being for enniatin B (51%). DON, mycophenolic acid and ZEA plus metabolites (α-zearalenol, β-zearalenol) were the most abundant mycotoxins.     

Utilising an LC-MS/MS-based multi-biomarker approach to assess mycotoxin exposure in the Bangkok metropolitan area and surrounding provinces

Human exposures to mycotoxins through dietary intake are a major health hazard and may result in various pathophysiological effects. Although Thailand is a country at increased risk due to its climatic conditions, no comprehensive dataset is available to perform proper exposure assessment of its population with regard to mycotoxins. Therefore, this pilot study was conducted to investigate and evaluate the exposure levels of major mycotoxins (aflatoxin B₁, ochratoxin A, fumonisins, zearalenone and trichothecenes). Sixty first-morning urine samples were collected from healthy volunteers who live in the Bangkok metropolitan area and surrounding provinces (Pathumthani, Nonthaburi, Samutprakarn and Samutsakorn). Urine samples were analysed by a LC-MS/MS-based multi-biomarker method following a so-called ‘dilute and shoot’ approach. Results generally indicated low mycotoxin exposures in most individuals through the determination of the four biomarkers that were detected in urine samples, i.e. aflatoxin M₁, ochratoxin A (OTA), as well as the deoxynivalenol (DON) metabolites DON-3-glucuronide and DON-15-glucuronide in 10 of 60 individuals. The maximum concentrations were used to estimate the daily intake confirming that none of the individuals exceeded the tolerable daily intake (TDI) of DON (maximum 26% of TDI) or OTA (maximum 22% of TDI). However, the maximum exposure of aflatoxin B₁, estimated to be 0.91 µg (kg bw)^–1 day^–1, should raise some concerns and suggests further studies utilising a more sensitive method. Low exposure to Fusarium toxins was also confirmed by the absence of zearalenone, α-zearalanol, β-zearalanol and zearalenone-14-glucuronide as well as T-2 toxin, HT-2 toxin, nivalenol and free DON. This is the first multi-mycotoxin biomarker study performed in Southeast Asia.     

Occurrence of Fusarium species and trichothecenes in Nigerian maize

A total of 180 maize samples meant for human consumption from four maize-producing states of southwestern Nigeria were screened for twelve major Fusarium mycotoxins (trichothecenes). Mycological examination of the samples showed that Fusarium verticillioides was the most commonly isolated fungi (71%), followed by F. sporotrichioides (64%), F. graminearum (32%), F. pallidoroseum (15%), F. compactum (12%), F. equiseti (9%), F. acuminatum (8%), F. subglutinans (4%) and F. oxysporum (1%). The trichothecenes include deoxynivalenol (DON), 3, mono-acetyldeoxynivalenol (3-AcDON), 15, mono-acetyldeoxynivalenol (15-AcDON), nivalenol (NIV), HT-2 toxin (HT-2), neosolaniol (NEO), T-2 toxin (T-2), T-2 tetraol and T-2 triol, diacetoxyscirpenol (DAS), MAS-monoacetoxyscirpenol (MAS) and fusarenone-X. Quantification was by high performance liquid chromatography coupled with mass spectroscopy (HPLC/MS); the detection limits for each of the mycotoxins varied between 20 and 200 microg kg(-1). Sixty six samples (36.3%) were contaminated with trichothecenes, DON (mean: 226.2 microg kg(-1); range: 9.6-745.1 microg kg(-1)), 3-AcDON (mean: 17.3 microg kg(-1); range: 0.7-72.4 microg kg(-1)) and DAS (mean: 16.0 microg kg(-1); range: 1.0-51.0 microg kg(-1)) were detected in 22%, 17% and 9% of total samples respectively. There were no 15-AcDON, NIV, HT-2, NEO, T-2, T-2 tetraol, T-2 triol, MAS and fusarenone-X detected. This is the first comprehensive report about the natural occurrence of DON, AcDON and DAS in maize for direct human consumption in Nigeria.     

A survey of free and conjugated deoxynivalenol in the 2008 corn crop in Ontario, Canada

BACKGROUND: Deoxynivalenol (DON, vomitoxin), one of the most important mycotoxins produced by many Fusarium species, is found as a common contaminant of crops worldwide. Recent studies have described the presence of conjugated forms of DON (glycosides and fatty acid). The aim of the current study was therefore to investigate the natural occurrence of free and conjugated DON in Canadian corn. RESULTS: Free and conjugated DON was determined by gas chromatography‐mass spectrometry (GC‐MS) and enzyme‐linked immunosorbent assay (ELISA) in 86 corn samples collected from the 2008 crop in Ontario, Canada. Free DON concentrations determined by ELISA were similar to values determined in most samples using GC‐MS. Conjugated DON was detected in 72 samples. Levels of free DON ranged from 0.17 to 14.00 µg g^?1 using GC‐MS. The highest levels of free DON were found in corn samples from the southern and southwestern regions of Ontario, while samples from eastern regions were less contaminated. Conjugated DON was found mainly in corn from the east‐central region, with five of six samples showing high levels of conjugated DON (up to 43% increase in DON following acid hydrolysis). Low levels of conjugated DON (≤10% increase in DON following acid hydrolysis) were detected in the majority of corn samples from the southwestern region (nine of 19 samples) and from the central region (16 of 36 samples). CONCLUSION: The current survey emphasizes the frequency of conjugated DON in Ontario grown corn and the potential challenges in understanding the hazard posed by DON‐contaminated foodstuffs and feedstuffs. Copyright © 2011 Society of Chemical Industry     

Mycotoxin co-occurrence in rice,oat flakes and wheat noodles used as staple foods in Ecuador

The co-occurrence of aflatoxin B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂), ochratoxin A (OTA), deoxynivalenol (DON), fumonisin B₁ (FB₁), zearalenone (ZEN), and HT-2 and T-2 toxins in the main Ecuadorian staple cereals (rice, oat flakes, and yellow and white wheat noodles) was evaluated. A ultra high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) method was developed and validated to screen for the presence of these mycotoxins in those cereal matrices. Matrix-matched calibration curves were used to compensate for ion suppression and extraction losses and the recovery values were in agreement with the minimum requirements of Regulation 401/2006/EC (70–110%). For most mycotoxins, the LODs obtained allowed detection in compliance with the maximum permitted levels set in Regulation EC/2006/1881, with the exception of OTA in all cereals and AFB1 in yellow noodles. Extra target analysis of OTA in oat flakes and wheat noodles was performed by HPLC with fluorescence detection. High rates of contamination were observed in paddy rice (23% DON, 23% FB₁, 7% AFB₁, 2% AFG₁ and 2% AFG₂), white wheat noodles (33% DON and 5% OTA) and oat flakes (17% DON, 2% OTA and 2% AFB₁), whereas the rates of contamination were lower in polished rice (2% AFG₁ and 4% HT-2 toxin) and yellow noodles (5% DON). Low rates of co-occurrence of several mycotoxins were observed only for white wheat noodles (5%) and paddy rice (7%). White noodles were contaminated with DON and/or OTA, while combinations of AFG₁, AFB₁, DON and FB₁ were found in paddy rice. Yellow noodles were contaminated with DON only; oat flakes contained DON, OTA or AFB₁, and polished rice was contaminated with AFG₁ and HT-2 toxin.     

Incidence of trichothecenes and zearalenone in poultry feed mixtures from Slovakia

A total of 50 samples of poultry feed mixtures of Slovakian origin were analyzed for eight toxicologically significant Fusarium mycotoxins, namely zearalenone (ZON), A-trichothecenes: diacetoxyscirpenol (DAS), T-2 toxin (T-2) and HT-2 toxin (HT-2) and B-trichothecenes: deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), 15-acetyl-deoxynivalenol (15-ADON) and nivalenol (NIV). The A-trichothecenes and the B-trichothecenes were detected by means of high pressure liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS) and gas chromatography electron capture detection (GC-ECD), respectively. Reversed phase-high performance liquid chromatography with a fluorescence detector (RP-HPLC-FLD) was used for ZON detection. The most frequent mycotoxin detected was T-2, which was found in 45 samples (90%) in relatively low concentrations ranging from 1 to 130 microg kg(-1) (average 13 microg kg(-1)), followed by ZON that was found in 44 samples (88%) in concentrations ranging from 3 to 86 microg kg(-1) (average 21 microg kg(-1)). HT-2 and DON were detected in 38 (76%) and 28 (56%) samples, respectively, in concentrations of 2 to 173 (average 18 microg kg(-1)) for HT-2 and 64 to 1230 microg kg(-1) sample (average 303 microg kg(-1)) for DON. The acetyl-derivatives of DON were in just four samples, while NIV was not detected in any of the samples investigated. In as many as 22 samples (44%), a combination of four simultaneously co-occurring mycotoxins, i.e. T-2, HT-2, ZON and DON, was revealed. Despite the limited number of samples investigated during this study poultry feed mixtures may represent a risk from a toxicological point of view and should be regarded as a potential source of the Fusarium mycotoxins in Central Europe. This is the first reported study dealing with zearalenone and trichothecene contamination of poultry mixed feeds from Slovakia.     

Determination of trichothecenes in cereals and cereal-based products by liquid chromatography–tandem mass spectrometry

A sensitive, accurate and precise method for the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in different food matrices, including wheat, maize, barley, cereal-based infant foods, snacks, biscuits and wafers, has been developed. The method, using liquid chromatography coupled with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC–APCI–MS/MS), allowed unambiguous identification of the selected trichothecenes at low µg per kg levels in such complex food matrices. A clean-up procedure, based on reversed phase SPE Oasis® HLB columns, was used, allowing good recoveries for all studied trichothecenes. In particular, NIV recoveries significantly improved compared to those obtained by using Mycosep® #227 columns for clean-up of the extracts. Limits of detection in the various investigated matrices ranged 2.5–4.0 µg kg^?1 for NIV, 2.8–5.3 µg kg^?1 for DON, 0.4–1.7 µg kg^?1 for HT-2 and 0.4–1.0 µg kg^?1 for T-2. Mean recovery values, obtained from cereals and cereal products spiked with NIV, DON, HT-2 and T-2 toxins at levels from 10 to 1000 µg kg^?1, ranged from 72 to 110% with mean relative standard deviation lower than 10%. A systematic investigation of matrix effects in different cereals and cereal products was also carried out by statistically comparing the slopes of standard calibration curve with matrix-matched calibration curve for each of the four toxins and the eight matrices tested. For seven of the eight matrices tested, statistically significant matrix effects were observed, indicating that, for accurate quantitative analysis, matrix-matched calibration was necessary. The method was applied to the analysis of 57 samples of ground wheat originated from South Italy and nine cereal food samples collected from retail markets.     

Occurrence of mycotoxins in maize,grass and wheat silage for dairy cattle in the Netherlands

The occurrence of mycotoxins in 140 maize silages, 120 grass silages and 30 wheat silages produced in the Netherlands between 2002 and 2004 was determined using a liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) multi-method. Deoxynivalenol (DON) was detected above the limit of quantification (LOQ) of 250 μg kg^?1 in 72% of maize and 10% of wheat silages. Average DON concentrations were 854 and 621 μg kg^?1, respectively, and maximum concentrations 3142 and 1165 μg kg^?1, respectively. Zearalenone was detected above the LOQ of 25 μg kg^?1 in 49% of maize and 6% of grass silages. Average zearalenone concentrations were 174 and 93 μg kg^?1, respectively, and maximum concentrations 943 and 308 μg kg^?1, respectively. The incidences and average concentrations of DON and zearalenone in maize silage were highest in 2004. The incidence of other mycotoxins was low: fumonisin B1 and 15-acetyl-DON were detected in 1.4 and 5% of maize silages, respectively, and roquefortin C in 0.8% of grass silages. None of the silages contained aflatoxins, ochratoxin A, T2-toxin, HT2-toxin, sterigmatocystin, diacetoxyscirpenol, fusarenon-X, ergotamine, penicillinic acid, or mycophenolic acid. This study demonstrates that maize silage is an important source of DON and zearalenone in the diet of dairy cattle. Since the carryover of these mycotoxins into milk is negligible, their occurrence in feed is not considered to be of significant concern with respect to the safety of dairy products for consumers. Potential implications for animal health are discussed.     

Determination of mycotoxins in biscuits,dried fruits and fruit jams: an assessment of human exposure

ABSTRACT

A reliable, fast and simple method using UHPLC-MS/MS was developed for the determination of aflatoxins B₁ (AFB1), G₁ (AFG1), B₂ (AFB2) and G₂ (AFG2), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEA), HT-2 toxin and T-2 toxin in crude extracts of biscuits with fruit filling, cookies, dried fruits and fruit jams. The method was successfully demonstrated on 39 samples of biscuits with fruit filling, 34 cookies, 14 dried fruits and 10 fruit jams. The mycotoxins detected in biscuits samples were ZEA, OTA, T-2 and AFB1 with an average concentrations of positive samples of 2.64, 4.10, 8.13 and 1.32 µg kg^?1, respectively; while the mycotoxins detected in jam samples were AFB1, OTA, T-2 and AFB2 with an average concentrations of positive samples of 2.00, 17.7, 4.37 and 1.15 µg kg^?1, respectively. The results showed that the majority of samples were in compliance with relevant regulations. However in eight samples of biscuits and three samples of fig jam the contents of OTA were higher than the existing OTA limits. The combined dietary exposure of selected mycotoxins was estimated for the first time for children, adolescents and adults. The estimated combined dietary exposures were all lower than the proposed value assumed to predict a possible risk scenario.     

Deoxynivalenol,zearalenone and T-2 in grain based swine feed in Hungary

Fusarium genera can produce trichothecenes like deoxynivalenol (DON), zearalenone (ZEN) and T-2 toxin, which can occur in feed cereal grains. Enzyme-linked immunosorbent assays (ELISA) tests of different Hungarian swine feedstuff proved that these mycotoxins were present. In this survey, 45 feed samples from 3 significant Hungarian swine feedstuff manufacturers were tested. ELISA methodology validation showed mean recovery rates in ranges from 85.3% to 98.1%, with intermediate precision of 86.9-96.9% and variation coefficients of 3.4–5.7% and 5.9–7.1%, respectively. The results showed that among Fusarium toxins, generally DON was present in the highest concentration, followed by T-2 and finally ZEN in all tested swine feeds. Each of the mycotoxins was found above the limit of detection in all swine feedstuffs. Boars feed’s DON (average ± standard deviation was 872 ± 139 µg kg^?1) and ZEN (172 ± 18 µg kg^?1) results of one of the manufacturers were above the guidance values. It indicates the necessity for efficient monitoring of DON, ZEN and T-2 mycotoxins in swine feeds.     

Determination of trichothecenes and zearalenones in grain cereal,flour and bread by liquid chromatography tandem mass spectrometry

Although analytical methods have been already reported for legislated mycotoxins as trichothecenes and zearalenone (ZON) separately, we describe the optimization of a simple and rapid multimycotoxin method for the determination of a total of 12 mycotoxins simultaneously, nine trichothecenes (NIV, DON, FUS-X, DAS, 15-AcDON, 3-AcDON, NEO, HT-2, T-2 T2), and zearalenone and its metabolites (ZON, α-ZOL, β-ZOL), of different origin (wheat, oat, barley and spelt) and in three different products where these substance can be present (grain, flour and bread) reach the food chain and cause toxic effect either in humans or animals. The extraction procedure was based on a mixture of acetonitrile/water (84/16, v/v), which provided the highest recoveries and the lowest matrix effect. DON-d1 was used as internal standard (I.S.) which helped to compensate the significant matrix effect observed for some matrices, and to obtain high success in the method validation and to reach the parameters compiled in Commission Decision, 2002/657/EC. Analytes were determinate by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Relative recoveries obtained were higher than 70% for the studied mycotoxins the four cereal. Good linearity (r² > 0.992) was obtained and quantification limits (2.5–25 ng/g) were below European regulatory levels. Repeatability, expressed as relative standard deviation, was always lower than 11%, whereas interday precision was lower than 11% for the developed method.     

Less Fusarium infestation and mycotoxin contamination in organic than in conventional cereals

A total of 602 samples of cereals, consisting of organically and conventionally produced barley, oats and wheat, were collected at harvest during 2002–2004 in Norway. Organic and conventional cereals were sampled in comparable numbers regarding cereal species, localisation and harvest time, and analysed for Fusarium mould and mycotoxins. Fusarium infestation and mycotoxin content were dependent on cereal species and varied year-by-year. However, in all cereal species, Fusarium infestation and levels of important mycotoxins were significantly lower when grown organically than conventionally. Concerning the most toxic trichothecenes, HT-2 and T-2 toxin, lower concentrations were found in organic oats and barley. Wheat was not contaminated by HT-2 and T-2, but lower concentrations of deoxynivalenol (DON) and moniliformin (MON) were found when organically produced. For mycotoxins considered to constitute the main risk to humans and animals in Norwegian cereals, i.e. HT-2 in oats and DON in oats and wheat, the median figures (mean levels in brackets) were as follows: HT-2 in organic and conventional oats were <20 (80) and 62 (117) µg/kg, DON in organic and conventional oats were 24 (114) and 36 (426) µg/kg, and DON in organic and conventional wheat were 29 (86) and 51 (170) µg/kg, respectively. Concentrations of HT-2 and T-2 in the samples were strongly correlated (r = 0.94). Other mycotoxins did not show a significant correlation to each other. Both HT-2 and T-2 concentrations were significantly correlated with infestation of F. langsethiae (r = 0.65 and r = 0.60, respectively). Concentrations of DON were significantly correlated with F. graminearum infestation (r = 0.61). Furthermore, nivalenol (NIV) was significantly correlated with infestation of F. poae (r = 0.55) and MON with F. avenaceum (r = 0.37). As lower Fusarium infestation and mycotoxin levels were found in organic cereals, factors related to agricultural practice may reduce the risk of contamination with Fusarium mycotoxins. Studies of these issues will be presented separately.     

High-Throughput Analytical Strategy Based on Modified QuEChERS Extraction and Dispersive Solid-Phase Extraction Clean-up Followed by Liquid Chromatography-Triple-Quadrupole Tandem Mass Spectrometry for Quantification of Multiclass Mycotoxins in Cereals

A simple, reliable, efficient, selective and sensitive QuEChERS-based (quick, easy, cheap, effective, rugged and safe) sample preparation strategy, involving an initial partitioning step using acidified acetonitrile (ACN), MgSO₄, NaCl and citrate buffer salts, combined with dispersive solid-phase extraction (d-SPE) clean-up, is proposed for the simultaneous multiclass mycotoxins quantification, including aflatoxins, ochratoxins, fumonisins, trichothecenes and zearalenone, in cereals. The final clear extracts were concentrated under vacuum to near dryness and taken-up with the initial mobile phase (MeOH:H₂O;70:30, v/v) previous to reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis. Careful optimization of the LC-ESI-MS/MS parameters was achieved in order to attain a fast separation and increased sensitivity. The detection was carried out on a triple-quadrupole tandem mass spectrometry (MS/MS) by electrospray ionization in positive ion mode (ESI⁺) with multiple reaction monitoring (MRM). Tandem MS conditions were optimised in order to increase selectivity, selecting the best transitions (parent ion to quantifier and qualifier ions) for quantification and identification. The performance of the method was assessed and compared to European Commission (EC) Regulations, by studying the selectivity, specificity, limits of detection (LOD) and quantification (LOQ), linear dynamic range (LDR), matrix effect, accuracy, precision, and uncertainty. Good linearity (r ²?>?0.9713) was achieved for all mycotoxins investigated, and LODs (S/N?=?3) and LOQs (S/N?=?10) were below the tolerance levels of mycotoxins set by EC. Recoveries of the extraction process, obtained with different spiked concentrations, ranged from 72.9 to 120.6 %, with relative standard deviations (RSD) lower than 23.0 %. Only in 6 % of all combinations did the RSD values exceed 15 %. Matrix effects were observed by comparing the slope of matrix-matched standard calibration with that of the solvent. The developed method was applied to evaluate the co-occurrence of multiclass mycotoxins in cereals collected at the importation points and consumer habitations at Madeira Island. Samples collected at importation points (15 wheat samples, 4 maize samples and 2 rice samples) showed the presence of DON in three wheat samples, and FB1 and HT-2 in one wheat sample. Three maize samples were detected with FB1 (two samples) and AFG₂ (one sample) whereas one rice sample was detected with ZEN. The results revealed the absence of target mycotoxins on the rice samples collected at consumer habitations. None of the studied cereal samples exceeded the maximum permissible limits or indicative levels set by the EC which means that the particular Madeira Island subtropical climate conditions do not represent a major risk for cereal contamination, taking into account the investigated mycotoxins.     

Effect of time, temperature and solvent on the stability of T-2 toxin, HT-2 toxin, deoxynivalenol and nivalenol calibrants

The influence of solvent, storage time and temperature on the stability of the trichothecene mycotoxins T-2 toxin (T-2), HT-2 toxin (HT-2), deoxynivalenol (DON) and nivalenol (NIV) was investigated. Toxins in acetonitrile, ethyl acetate or as thin film were stored in sealed glass ampoules at -18, 4, 25 and 40°C for up to 24 months. Samples were analysed by HPLC with UV detection. The results show that acetonitrile was the preferred solvent and no significant ( t 0.95 -test) decomposition occurred for any of the four trichothecenes when stored for 24 months at 25°C or 3 months at 40°C. T-2 and HT-2 in ethyl acetate or as thin film were also stable under the same conditions. DON and NIV in ethyl acetate or as thin film were stable for up to 24 months at -18°C, but a significant decomposition of DON and NIV in ethyl acetate was observed for both toxins after 24 months of storage at 4°C and after 12 months at 25°C. When stored as thin film, a significant trend of decomposition of DON occurred after 24 months of storage at 4°C and after 6 months of storage at 25°C. A significant decrease of NIV stored as thin film was observed after 9 months at 25°C. In conclusion, acetonitrile was the most suitable solvent for long-term storage of T-2, HT-2, DON and NIV.