Determination of deoxynivalenol in processed foods

Deoxynivalenol (DON), commonly referred to as vomitoxin, belongs to a class of naturally occurring mycotoxins produced by Fusarium fungi. The presence of DON in foods is a human health concern. The frequency of occurrence of DON in wheat is high, although cleaning prior to milling can reduce DON concentration in final products, and food processing can partially degrade the toxin. This paper describes a method for the determination of DON in some major wheat food products, including bread, breakfast cereals, pasta, pretzels, and crackers. Test samples containing 5% polyethylene glycol were extracted with water. After blending and centrifuging, the supernatant was diluted with water and filtered through glass microfiber filter paper. The filtrate was then passed through an immunoaffinity column and the toxins eluted with methanol. The toxins were then subjected to RPLC separation and UV detection. The accuracy and repeatability characteristics of the method were determined. Recoveries of DON spiked at levels from 0.5 to 1.5 microg/g in the five processed foods were >70%. SD and RSD values ranged from 2.0 to 23.5% and from 2.0 to 23.2%, respectively. HorRat values were <2 for all of the matrixes examined. The method was found to be acceptable for the matrixes examined. LC/MS/MS with multiple-reaction monitoring was used to confirm the identity of DON in naturally contaminated test samples.     

Determination of nivalenol and deoxynivalenol by liquid chromatography/atmospheric pressure photoionization mass spectrometry

Fusarium species, a plant pathogenic fungus of wheat and other cereals, produces toxic metabolites such as nivalenol (NIV) and deoxynivalenol (DON). Control of contamination by these toxins is very difficult, and a continuous survey of the occurrence is necessary for these toxins. Thus, the accurate and convenient determination of the cereals contaminated with these toxins is important for the supply of safe foods. A selective analytical method based on high‐performance liquid chromatography, combined with atmospheric pressure photoionization (APPI) mass spectrometry, has been developed for simultaneous determination of NIV and DON. The parameters investigated for the optimization of APPI were the ion source parameters fragmentor voltage, capillary voltage, and vaporizer temperature, and also mobile phase composition and flow rate. Furthermore, chemical noise and signal suppression of analyte signals due to sample matrix interference were investigated for APPI. The results indicated that APPI provides lower matrix effect and the correlation coefficient of NIV and DON in the range 0.2–100 ng · mL^?1 was above 0.999. Recoveries of NIV and DON in wheat ranged from 86 to 107% and limits of detection of NIV and DON were 0.20 ng · g^?1 and 0.39 ng · g^?1, respectively. In addition, the proposed method was applied for the analysis of naturally contaminated wheat samples. APPI was found to offer lower matrix effect and was a convenient technique for routine analysis of NIV and DON residues in wheat at trace levels. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of type A and type B trichothecenes in paprika and chili pepper using LC-triple quadrupole-MS and GC-ECD

There is a need to develop sensitive and accurate analytical methods for determining deoxynivalenol (DON), HT-2 toxin and T-2 toxin in paprika to properly assess the relevant risk of human exposure. An optimized analytical method for determination of HT-2 toxin and T-2 toxin using capillary gas chromatography with electron capture detection and another method for determination of DON by liquid chromatography-mass spectrometry in paprika was developed. The method for determination of HT-2 toxin and T-2 toxin that gave the best recoveries involved extraction of the sample with acetonitrile-water (84:16, v/v), clean-up by solid-phase extraction on a cartridge made of different sorbent materials followed by a further clean-up in immunoaffinity column that was specific for the two toxins. The solvent was changed and the eluate was derivatized with pentafluoropropionic anhydride and injected into the GC system. The limits of detection (LOD) for T-2 and HT-2 toxins were 7 and 3 μg/kg, respectively, and the recovery rates for paprika spiked with 1000 μg toxin/kg were 71.1% and 80.1% for HT-2 and T-2 toxins, respectively. For DON determination, the optimized method consisted of extraction with acetonitrile-water (84:16, v/v) solution followed by a solid-phase extraction clean-up process in a cartridge made of different sorbent compounds. After solvent evaporation in N₂ stream, the residue was dissolved and DON was separated and determined by LC-MS/MS. The LOD for this method was 14 μg DON/kg paprika sample and the DON recovery rate was 86.8%.     

农产品中脱氧雪腐镰刀菌烯醇的表面增强拉曼检测

利用便携式拉曼光谱仪建立了一个快速筛查与检测谷物中真菌毒素脱氧雪腐镰刀菌烯醇(DON)的表面增强拉曼散射(SERS)方法。首先利用实验室前期开发的方法制备了具有高活性的水凝胶SERS芯片。该SERS芯片是将预先制备的高SERS活性的单层碳基点(CDs)包裹的银纳米颗粒团聚体(a-AgNPs/CDs)与聚乙烯醇(PVA)水溶液混合均匀后,再利用循环冷冻-解冻的物理交联法制备而成的。实验优化了影响水凝胶SERS芯片对DON的SERS响应的实验条件,包括溶剂、浸泡温度和浸泡时间。在最佳的SERS检测条件下(溶剂为水-乙醇(1:1, v/v),浸泡温度为40 ℃,浸泡时间为5 min), DON的线性响应范围为1~10000 μg/kg(相关系数(R²)=0.9967),检出限(LOD)为0.14 μg/kg,表明该SERS基底具有较高的灵敏度。得益于水凝胶特殊的孔径结构,实际样品基质中常见的糖、蛋白质、油脂、色素等干扰物质都被阻隔在水凝胶外。因此,在复杂样品检测中仅需要简单的提取,而不需要复杂的分离处理。将该方法用于小麦粉中DON的检测,所得回收率为97.3%~103%,相对标准偏差为4.2%~5.0%。实验结果表明所建立的检测DON的SERS方法具有响应范围宽、灵敏度高、重复性好、响应迅速、操作简单、抗干扰能力强等优点,这说明本实验室所构建的水凝胶SERS芯片在粮食中生物毒素的快速筛查与检测方面具有良好的应用潜力。     

Rapid detection of nivalenol and deoxynivalenol in wheat using surface plasmon resonance immunoassay

A surface plasmon resonance (SPR) immunoassay using a monoclonal antibody was developed to measure nivalenol (NIV) and deoxynivalenol (DON) contamination in wheat. A highly sensitive and stable DON-immobilized sensor chip was prepared, and an SPR detection procedure was developed. The competitive inhibition assay used a monoclonal antibody that cross-reacts with NIV and DON. The half maximal inhibitory concentration (IC₅₀) values of the SPR assay were 28.8 and 14.9 ng mL⁻¹ for NIV and DON, respectively. The combined responses of NIV and DON in wheat were obtained using a simultaneous detection assay in a one-step cleanup procedure. NIV and DON were separated using a commercial DON-specific immunoaffinity column (IAC) and their responses were obtained using an independent detection assay. Spiked tests using these toxins revealed that recoveries were in the range 91.5-107% with good relative standard deviations (RSDs) (0.40-4.1%) and that detection limits were 0.1 and 0.05 mg kg⁻¹ for NIV and DON, respectively. The independent detection using IAC showed detection limits of 0.2 and 0.1 mg kg⁻¹ for NIV and DON, respectively. SPR analysis results were correlated with those obtained using a conventional LC/MS/MS method for wheat co-contaminated with NIV and DON. These results suggested that the developed SPR assay is a practical method to rapidly screen the NIV and DON co-contamination of wheat and one of a very few immunoassays to detect NIV directly.     

Use of the modified quick easy cheap effective rugged and safe sample preparation approach for the simultaneous analysis of type A- and B-trichothecenes in wheat flour

A suitable extraction and purification method for the simultaneous liquid chromatography–mass spectrometry (LC–MS) determination of five mycotoxins, three type A, diacetoxyscirpenol (DAS), T-2 toxin (T-2) and HT-2 toxin (HT-2), and two type B-trichothecenes, deoxynivalenol (DON) and nivalenol (NIV), has been optimised using a modified “Quick Easy Cheap Effective Rugged and Safe” (QuEChERS) method. Different solvents were studied in the extraction procedure to obtain better recoveries, which ranged from 86 to 108%, using a 85/15 (v/v) mixture of methanol/acetonitrile. The values obtained for recovery, repeatability and reproducibility of the optimized method are in agreement with Commission Directive 2005/26/EC for methods of analysis of Fusarium toxins. Finally, this optimized procedure was applied in wheat flour samples commercialized in Spain.     

Purification of deoxynivalenol from Fusarium graminearum rice culture and mouldy corn by high-speed counter-current chromatography

Deoxynivalenol (DON) is a mycotoxin produced by several Fusarium species and is toxic to a wide range of organisms, including human beings and livestock. To produce large amounts of pure DON for research purposes, a novel method using high-speed counter-current chromatography (HSCCC) was developed. Rice cultured with Fusarium graminearum and field mouldy corn infected by F. graminearum were extracted with methanol and found to contain 1.16 and 1.30 mg DON/g, respectively. The extracts were concentrated and then separated using a biphasic solvent system consisting of ethyl acetate-water (1:1, v/v). Collected fractions were analyzed by high-performance liquid chromatography (HPLC) and identified by congruent retention time and UV/vis spectrum and mass spectrometric data. Fractions containing DON were combined and freeze-dried. This method produced 116 mg and 65 mg DON with a purity of greater than 94.9% from 200 g of the rice culture and the mouldy corn, respectively. The HSCCC method had a recovery rate of DON at 88% from the crude extracts of both samples. This one-step purification method provided a simple and effective tool for obtaining a large amount of DON, an essential material for studies related to toxicology and detoxification of this mycotoxin.     

Analysis of T-2 and HT-2 toxins in cereal grains by immunoaffinity clean-up and liquid chromatography with fluorescence detection

A sensitive, precise and accurate method has been developed for the simultaneous determination of T-2 and HT-2 toxins in cereal grains at ppb levels using high-performance liquid chromatography (HPLC) with fluorescence detection and 1-antroylnitrile (1-AN) as labeling reagent after immunoaffinity clean-up. Cereal samples were extracted with methanol/water (90:10, v/v), and the extracts were cleaned-up through commercially available immunoaffinity columns containing monoclonal anti-T-2 antibodies (T-2 test HPLC, Vicam). T-2 and HT-2 toxins were quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 381 nm, emission wavelength 470 nm) after derivatization with 1-AN. The monoclonal antibody showed 100% cross-reactivity with both T-2 and HT-2 toxin, and the immunoaffinity column clean-up was effective up to 1.4 microg of both toxins. The method was successfully applied to the analysis of T-2 and HT-2 toxins in wheat, maize and barley. Recoveries from spiked samples with toxin levels from 25 to 500 microg/kg ranged from 70% to 100%, with relative standard deviation generally lower than 8%. The limit of detection of the method was 5 microg/kg for T-2 toxin and 3 microg/kg for HT-2 toxin, based on a signal-to-noise ratio 3:1. HT-2 toxin was detected in ten naturally contaminated wheat samples out of 14 samples analyzed, with toxin levels ranging from 10 to 71 microg/kg; three of them contained also T-2 toxin up to 12 microg/kg.     

Analysis of some metabolites of T-2 toxin, diacetoxyscirpenol and deoxynivalenol by thermospray high-performance liquid chromatography-mass spectrometry

Detection and identification of mycotoxin metabolites is a very challenging task. In order to achieve adequate sensitivity and specificity an analytical technique must overcome serious matrix interferences. Gas chromatography-mass spectrometry (GC-MS) which has the sensitivity and specificity to detect and identify mycotoxin metabolites requires hydrolysis of conjugated metabolites as well as derivatization. Thermospray high-performance liquid chromatography-mass spectrometry (HPLC-MS) offers the sensitivity, specificity, and structural information to detect and identify some mycotoxin metabolites in fecal and urine samples without derivatization. The mycotoxins evaluated in this study include deoxynivalenol (DON), T-2 toxin, and diacetoxyscirpenol. The de-epoxy and hydroxy metabolites of each toxin and the glucuronide conjugate of DON were isolated, extracted, and analyzed to detect their occurrence in animals. The thermospray mass spectra of the toxins showed an [M + H]+ ion and numerous structurally significant fragment ions in the positive ion detection mode. Negative ion detection exhibited primarily [M + acetate]- cluster ions with less fragmentation than observed by positive ion detection. The operation of the interface in the filament-on mode greatly increased the sensitivity in both positive and negative ion detection mode. Detection limits of 50-500 pg injected on column are obtained for these toxins and their metabolites using multiple ion detection. The urine and fecal extracts from rats, hens, and cows did not interfere with the HPLC-MS analysis for the specific metabolites or the glucuronide conjugate.     

Use of a multifunctional column for the determination of deoxynivalenol in grains, grain products, and processed foods

Deoxynivalenol (DON), also known as vomitoxin, belongs to a class of naturally occurring mycotoxins produced by Fusarium spp. DON, 12, 13-epoxy-3,7 trihydroxytrichothec-9-en-8-one, is one of the most frequently detected mycotoxins in agricultural commodities worldwide. A method consisting of extraction, filtration, column cleanup, and RP-HPLC-UV separation and quantitation was validated for the determination of DON in grains (rice and barley), grain products (whole wheat flour, white flour, wheat germ, and wheat bran), and processed foods (bread, breakfast cereals, and pretzels). A 25 g test portion was extracted with 100 mL acetonitrile-water (84 + 16, v/v). After blending for 3 min, the supernatant was applied to a multifunctional column (MycoSep 225). The purified filtrate (2 mL) was evaporated to dryness and redissolved in the mobile phase. The toxins were then subjected to RP-HPLC-UV analysis. The accuracy and repeatability characteristics of the method were determined. Recoveries of DON added at levels ranging from 0.5 to 1.5 microg/g for all test matrixes were from 75 to 98%. SD and RSD(r) ranged from 0.7 to 11.6% and 0.9 to 12.7%, respectively. Within-laboratory HorRat values were from 0.1 to 0.7 for all matrixes analyzed. The method was found to meet AOAC method performance criteria for grains, grain products, and processed foods. The identity of DON in naturally contaminated test sample extracts was confirmed by HPLC/MS/MS analysis.     

Determination of trichothecenes in cereals by gas chromatography with ion trap detection

Summary A method for determination of the trichothecene toxins, deoxynivalenol, 3α-acetyl-deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin and diacetoxyscirpenol in cereals (wheat, barley, oats, corn) is described. Extraction was performed according to Tanaka et al. (J. Chromatogr.328, 271 (1985)) [33], derivatization by trifluoroacylation with trifluoroacetic acid anhydride. For quantitation and confirmation a capillary gas chromatograph combined with a selective mass detector (ion trap) working in CI-mode with methanol as reagent gas was used. The quantitation limit for the complete method is 1–5 μg/kg, depending on the chemical characteristics of each toxin and cleanness of the extracts. Recoverics from spiked cereals were 78–89%.     

Triazole Fungicide Residues and Their Inhibitory Effect on Some Trichothecenes Mycotoxin Excretion in Wheat Grains

Wheat is one of the global strategic crops and ranks third in terms of cereals production. Wheat crops are exposed to many fungal infections during their cultivation stages, some of which have the ability to secrete a number of toxic secondary metabolites that threaten the quality of the grains, consumer health, producer economics, and global trade exchange. Fifty-four random samples were collected from wheat which originated from different countries. The samples included 14 types of soft wheat to study the extent of their contamination with deoxynivalenol (DON) and T-2 toxin by auto-ELISA technology and r-biopharm microtiter plate. All samples were contaminated with DON toxin except one sample, and the values ranged between 40.7 and 1018.8 µg/kg⁻¹. The highest contamination rates were in Lithuanian wheat and the lowest was in Indian wheat. Meanwhile, the highest average level of T-2 toxin contamination was in Lithuanian wheat grains with 377.4 µg/kg⁻¹, and the lowest average was 115.3 µg/kg⁻¹ in Polish wheat. GC-MS/MS and multiple reaction monitoring mode (MRM) were used to detect 15 triazole derivatives in the collected samples, which may be used to combat fungal diseases on wheat during the growing season. Only 9 derivatives were found: simeconazole, penconazole, hexaconazole, cyproconazole, diniconazole, tebuconazole, metconazole, fenbuconazole, and difenoconazole. These derivatives varied according to the origin of the wheat samples as well as their concentration, whereas another 6 derivatives were not detected in any samples. A direct inverse relationship was found between the DON concentration in the samples and the residues of simeconazole, penconazole, diniconazole, tebuconazole, metconazole, fenbuconazole, and difenoconazole, and the T-2 toxin showed the same relationship except for tebuconazole. The safe and rational use of some triazole derivatives may be a new approach and a promising strategy to not only reduce plant diseases and their problems, but also to get rid of some mycotoxins as grain contaminants.     

Development and validation of a liquid chromatography/atmospheric pressure photoionization-tandem mass spectrometric method for the analysis of mycotoxins subjected to commission regulation (EC) No. 1881/2006 In cereals

A sensitive and reliable liquid chromatography/photoionization (APPI) tandem mass spectrometry method has been developed for determining nine selected mycotoxins in wheat and maize samples. The analytes were chosen on the basis of the mycotoxins under EU Commission Regulation (EC) No. 1881/2006, i.e., deoxynivalenol (DON), zearalenone (ZON), aflatoxins (AFs), and ochratoxin A (OTA), and considering the possibility of a near future regulation for T-2 and HT-2 toxins. Mycotoxins were extracted from samples by means of an one-step solvent extraction without any cleanup. The developed multi-mycotoxin method permits simultaneous, simple, and rapid determination of several co-existing toxins separated in a single chromatographic run, in which AFs, T-2 and HT-2 toxin are acquired in positive, while OTA, DON and ZON in negative mode. Although a moderate signal suppression was noticeable, matrix effect did not give significant differences at p = 0.05. Then, calibration in standard solution were used for quantitation. Based on the EU Commission Decision 2002/657/EC, the method was in-house validated in terms of ruggedness, specificity, linearity, trueness, within-laboratory reproducibility, decision limit (CCα) and detection capability (CCβ). For all the analytes, the regression coefficient r ranged between 0.8752 (DON in wheat) and 0.9465 (ZON in maize), biases related to mean concentrations were from −13% to +12% of the nominal spiking level, and the overall within-laboratory reproducibility ranged 3–16%; finally, CCα values did not differ more than 20% and CCβ not more than 42% from their respective maximum limit. Method quantification limits ranged from 1/20 (AFG1) to 1/4 (AFG2 and OTA) the maximum limit established by European Union in the Commission Regulation (EC) No. 1881/2006 and its subsequent amendments.     

Co-isolation of deoxynivalenol and zearalenone with sol–gel immunoaffinity columns for their determination in wheat and wheat products

The paper describes a sample clean-up method for the co-isolation of deoxynivalenol (DON) and zearalenone (ZON), two mycotoxins naturally co-occurring in wheat. The method is based on immunoaffinity columns prepared by co-immobilising anti-DON and anti-ZON antibodies in a porous sol–gel glass. The main task in developing the method consisted in finding a loading medium allowing retention of both analytes as well as a common elution medium for the dissociation of both antigen–antibody complexes formed. This can be achieved by co-extracting DON and ZON with ACN–water (60:40, v/v), reducing the acetonitril concentration to 2.5% before loading an aliquot of the diluted sample extract onto the DON/ZON column. The columns are washed with 5 ml of MeOH–water (10:90, v/v) before DON and ZON are co-eluted with 4 ml of ACN–water (50:50, v/v). Concentrations of DON and ZON are determined with HPLC-UV and HPLC-fluorescence detection, respectively. The sample clean-up method was shown to be applicable to wheat and wheat products, e.g., cornflakes, milk wheat mash and rusk. Spiking experiments (spike level 500 μg DON/kg and 50 μg ZON/kg) resulted in recovery rates from 82% to 111%.     

Determination of Fusarium mycotoxins by liquid chromatography/tandem mass spectrometry coupled with immunoaffinity extraction

A method for the simultaneous quantitative determination of deoxynivalenol (DON), T‐2 toxin (T‐2), HT‐2 toxin (HT‐2) and zearalenone (ZEN) in wheat and biscuit by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) coupled with immunoaffinity extraction is described. A clean‐up was carried out using a DZT MS‐PREP® immunoaffinity column (IAC), and the effect of the sample dilution rate and sample loading was investigated. Furthermore, the effects of ion suppression of a multifunctional column (MFC) and the IAC in the clean‐up were compared. The results with the DZT MS‐PREP® IAC showed that it is possible to make the sample dilution rate low, and indicated a higher solvent‐tolerance than usual with an IAC. Sample loading was optimized at 0.25 g. Ion suppression was lowered by purification of the toxins using the DZT MS‐PREP® IAC. Recoveries of each mycotoxin from wheat and biscuit samples spiked at two levels ranged from 78 to 109%. The limits of detection in wheat and biscuit was in the range of 0.03–0.33 ng·g^?1. From these studies, it is suggested that use of an IAC is effective in the clean‐up of each mycotoxin, and, when combined with LC/ESI‐MS/MS, it is good for the determination of mycotoxins in foodstuffs due to its rapidity and high sensitivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Inhibition of mycotoxin deoxynivalenol generation by using selenized glucose

Selenized glucose can be easily prepared via the selenization reaction of glucose using in situ generated NaHSe as the selenization reagent. The technique has been industrialized to produce the chemical in kilogram scale, making it an easily available material in laboratory presently. The selenized glucose may be widely used as the starting material for the preparation of selenium-containing catalysts, as the organoselenium additive for feeds, and as the efficient selenium-enriched foliar fertilizers. In this work, we found that treating Fusarium graminearum, a fungal pathogen inciting wheat scab disease, with selenium glucose could significantly inhibit the generation of the deoxynivalenol (DON) toxin, which might be a breakthrough for reducing the detriment of the wheat scab disease.     

Quantification of deoxynivalenol in wheat using an immunoaffinity column and liquid chromatography

A simple and accurate method to quantify the mycotoxin deoxynivalenol (DON) in wheat is described. The method uses immunoaffinity chromatography for DON isolation and liquid chromatography (LC) for toxin detection and quantification. Wheat samples are extracted in water, filtered twice and applied to an immunoaffinity column. Following a water wash, DON is eluted from the column with methanol and injected onto an LC system with a UV detector for quantification. Test performance was evaluated in terms of antibody specificity, limit of detection, percentage recovery, precision, column capacity, assay linearity and comparison with the GC-electron-capture detection (ECD) method of Tacke and Casper. Specificity of the immunoaffinity column cleanup procedure was confirmed with only DON (>80%) and its 15-C derivatives (40-50%) being recognized by the antibody while 3-C DON derivatives, nivalenol, T-2 and fusarenon-X did not bind. The limit of detection is at least 0.10 microg/g. Percentage recovery for the entire assay range averages 90% with an average relative standard deviation of 8.3%. Naturally contaminated samples showed comparable precision. Column capacity was determined to be 3.3 microg. The assay showed a high degree of linearity (r2=0.999) and an optimum assay range of 0.10 to 10.0 microg/g. Comparative analysis of 28 naturally or artificially contaminated wheat samples using DONtest-HPLC and the GC-ECD method of Tacke and Casper showed that DONtest-HPLC is a statistically significant predictor of the GC-ECD method (r2=0.982).     

Interlaboratory comparison study for the determination of the Fusarium mycotoxins deoxynivalenol in wheat and zearalenone in maize using different methods

Seventeen laboratories from six different countries, using their usual in-house methods, participated in an interlaboratory comparison test for the determination of the Fusarium mycotoxins deoxynivalenol (DON) in wheat and zearalenone (ZON) in maize. The toxins generally were extracted from maize and wheat employing mixtures of water, acidified water with an organic solvent or even pure water (for DON). While participants who used enzyme linked immuno sorbent assays (ELISA) for the determination of DON did not perform any clean-up, various techniques were applied for the purification of raw extracts (e.g. liquid/liquid extraction, solid phase extraction (SPE), immuno affinity chromatography (IAC)). For the final separation/quantification step either high performance liquid chromatography (HPLC) (mostly for ZON), gas chromatography (GC) (for DON) or ELISA were employed by participants. The aim of this study was to obtain information about the state of the art of mycotoxin analysis in cereals and to support a knowledge and experience exchange between the participating laboratories in the field of mycotoxin analysis. For each mycotoxin 5 different sample types were distributed, standard solutions (10.10 μg/ml ZON in methanol, 10.09 μg/ml DON in ethyl acetate), blank materials, spiked samples (75.1 μg/kg and 378.3 μg/kg ZON in maize, 126.2 μg/kg and 2519 μg/kg DON in wheat) and naturally contaminated maize and wheat. Coefficients of variation (CV) between laboratory mean results (outliers excluded) ranged from 6.2 to 27.7% for ZON and from 18.9 to 30.0% for DON. Except for the maize samples spiked at 75.1 μg/kg ZON the overall means (outliers rejected) statistically could not be distinguished from the respective target values. Average recoveries of the reported results ranged from 87.7 to 96.2% for ZON and from 94.2 to 108.5% for DON.     

Enzymatic hydrolysis of T-2 toxin for the quantitative determination of total T-2 and HT-2 toxins in cereals

The selective enzymatic deacetylation of T-2 toxin to give HT-2 toxin has been investigated in aqueous crude extracts of different cereals and exploited to develop an analytical method for the determination of the sum of T-2 and HT-2 toxins. The method has been validated for the analysis of total T-2 and HT-2 toxins in maize, wheat, and oats, showing recoveries from 72 to 97% for maize, from 67 to 84% for wheat, and from 61% to 87% for oats, at spiking levels of 20–400 μg/kg, with relative standard deviation lower than 10%. Liquid chromatography-tandem mass spectrometry was used for quantitative toxin determination. The potential biological role of this enzymatic conversion and its perspectives for application in the development of antibody-based analytical techniques are discussed.      

Simultaneous determination of several mycotoxins by rapid immunofiltration assay

A method is developed for the simultaneous rapid determination of three mycotoxins, zearalenone (ZEN), ochratoxin A (OTA), and fumonisin B1 (Fum) by membrane immunofiltration analysis using a marker enzyme of alkaline phosphatase (AP) and two mycotoxins, deoxynivalenol (DON) and total T-2 toxin (T2) and HT-2 toxin (HT2) with horseradish peroxidase (HRP). The analysis is based on a competitive interaction between the antigene, free and bound to the enzyme, and antibodies immobilized on a membrane. The procedures of membrane fabrication and the conditions of mycotoxin to determination in model mixtures and extracts from wheat, corn, and silage are optimized. The influence of sample preparation on the results of analysis is studied. It is shown that the additives of polymers favor the reduction of the matrix effect in the analysis of complex matrixes using conjugated HRP. The methods developed allow the determination of mycotoxins at a level of the maximal permissible concentrations legislated by EU directives. The corresponding values (μg/kg) are 50, 2.5, and 500 in wheat; 100, 2.5, and 500 in corn; and 125, 25, and 1250 in silage for the simultaneous quantification of ZEN, OTA, and Fum (AP marker). For the determination of DON and total T2/HT2 with HRP, 1250 (1000) and 100 (500) in wheat and corn(silage). The procedures were validated by the analysis of spiked and naturally contaminated samples. The analysis of 10 samples takes 25 min.