Mycotoxins In Cereals Research Articles

Rapid detection in food and feed

Chemical contaminant monitoring in food and feed is a very relevant issue in global food quality and safety according to ongoing and previous Rapid Alert System on Food and Feed (RASFF) alerts. Also, consumers have placed chemical contaminants at the top of the “worry scale” of food-related risks. The European Food Safety Authority urgently needs reliable exposure and toxicity data for many (emerging) contaminants such as perfluorinated compounds and plant and marine toxins. According to Regulation (EC) No. 178/2002, the food and feed industries are responsible for the safety of their products. In addition, Regulation (EC) No. 882/2004 requires that official controls are conducted, taking into account identified risks. Therefore, the demand for simplified and rapid test methods at critical control points in the entire chain has never been greater. At the same time, such novel screening tools should have multianalyte, multiclass (multiplex) capability by detecting as many contaminant parameters in parallel as possible within the shortest period of time. This topical collection on rapid detection in food and feed addresses this urgent demand for rapid multianalyte testing. The articles collected in this issue reflect the latest state of the art and the current and future challenges in rapid detection in food and feed. On the one hand, these articles include novel methods for the rapid quantification of food and feed contaminants by using established high-performance analytical techniques such as liquid chromatography–tandem mass spectrometry. On the other hand, major emphasis is given to innovative methods such as near-infrared hyperspectral imaging, quantum-dot-loaded liposomes for ultrasensitive onsite determination and easy-to-use multiplex dipstick assays. The methods presented have been developed and in many cases validated through interlaboratory comparison for a wide range of (emerging) contaminants in food and feed. These include mycotoxins in cereals and milk products, antibiotics in poultry muscle and in honey, brominated flame retardants in fish and inorganic arsenic in rice. We would like to express our great gratitude to the contributors; thanks to them this issue provides updated and high-quality original contributions on new developments in rapid detection in food and feed, its emerging applications and a picture of future trends. We hope that the collection of these articles stimulates further research to develop simplified and rapid test methods for the determination of contaminants in the entire food chain as an important means to protect consumers. Last but not least, we are also very thankful to the editorial team and the reviewers of Analytical and Bioanalytical Chemistry for their support and fantastic cooperation.

Automated regenerable microarray-based immunoassay for rapid parallel quantification of mycotoxins in cereals

An automated flow-through multi-mycotoxin immunoassay using the stand-alone Munich Chip Reader 3 platform and reusable biochips was developed and evaluated. This technology combines a unique microarray, prepared by covalent immobilization of target analytes or derivatives on diamino-poly(ethylene glycol) functionalized glass slides, with a dedicated chemiluminescence readout by a CCD camera. In a first stage, we aimed for the parallel detection of aflatoxins, ochratoxin A, deoxynivalenol, and fumonisins in cereal samples in a competitive indirect immunoassay format. The method combines sample extraction with methanol/water (80:20, v/v), extract filtration and dilution, and immunodetection using horseradish peroxidase-labeled anti-mouse IgG antibodies. The total analysis time, including extraction, extract dilution, measurement, and surface regeneration, was 19 min. The prepared microarray chip was reusable for at least 50 times. Oat extract revealed itself as a representative sample matrix for preparation of mycotoxin standards and determination of different types of cereals such as oat, wheat, rye, and maize polenta at relevant concentrations according to the European Commission regulation. The recovery rates of fortified samples in different matrices, with 55-80 and 58-79%, were lower for the better water-soluble fumonisin B1 and deoxynivalenol and with 127-132 and 82-120% higher for the more unpolar aflatoxins and ochratoxin A, respectively. Finally, the results of wheat samples which were naturally contaminated with deoxynivalenol were critically compared in an interlaboratory comparison with data obtained from microtiter plate ELISA, aokinmycontrol® method, and liquid chromatography-mass spectrometry and found to be in good agreement.

Alternaria mycotoxins in wheat – A 10 years survey in the Northeast of Germany

Although, the genus Alternaria is considered to be an important plant pathogen and capable to produce mycotoxins, the presence of Alternaria mycotoxins in cereals has been largely ignored both in Europe and overseas. This is very likely due to a lack of survey data. This study aimed at filling this gap by investigating a total of 1064 freshly harvested winter wheat samples from commercial farms in different regions of the State of Brandenburg (Germany) in the years from 2001 to 2010. We analysed alternariol (AOH), its monomethyl ether (AME), altenuene (ALT, since 2006) and tenuazonic acid (TeA) by a HPLC method with diode array and fluorescence detection. The most frequently found A. mycotoxin was TeA. An amount of 322 out of 1064 samples (30.3%) were naturally contaminated by TeA, 86 out of 1064 by AOH (8.1%), 33 out of 1064 by AME (3.1%) and 7 out of 267 samples (2.6%) were contaminated by ALT. The maximum toxin contents in all years were 4224 μg TeA kg−1, 832 μg AOH kg−1, 905 μg AME kg−1 and 197 μg ALT kg−1. A co-occurrence of several A. mycotoxins in wheat samples was infrequent: only three samples were contaminated by all the four toxins, 14 by three toxins, 61 by two toxins and 273 samples by only one toxin. The contamination of wheat ears in the State of Brandenburg in the wet years 2010, 2009 and 2002 were most pronounced, whereas 2001 and 2008 were A. “toxin free” years. The accumulation of TeA in freshly harvested wheat kernels seems to depend on preceding crop and tillage. Minimum tillage practices and maize as well as winter wheat as preceding crops led to increased TeA concentrations in wheat. A co-occurrence of mycotoxins produced by several mycotoxigenic fungi, e.g. Fusarium and Alternaria spp., is discussed.

Experimental design for in-house validation of a screening immunoassay kit. The case of a multiplex dipstick for Fusarium mycotoxins in cereals

The benefits of using rapid qualitative methods to verify compliance of food and feed with legislation requirements include user-friendly format, the possibility of detection without expensive instrumentation, rapid response and affordable price. Prior to their use, however, the methods have to pass validation experiments, in order to assess their performance profile. An experimental protocol for in-house validation of a screening immunoassay has been designed and applied to evaluate performance characteristics of a multiplex dipstick kit for the determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat and maize. The test is intended for screening of cereals on the presence/absence of these mycotoxins at maximum permitted levels established by European legislation or target levels. The response of the measurement is determined with a reader device. Samples classified as negative are considered as compliant, whereas positive samples need to be re-analysed with confirmatory methods. The in-house validation design consisted of three steps, namely (1) estimating the precision of the method including "between day" effects and influences from different varieties of the matrices, (2) establishing robust cutoff values for the dipstick response at target mycotoxin levels assuming an acceptable rate of false negative results of 5% and (3) assessment of the rate of false positive results of blank samples and samples containing the target analytes below the legal limits. The total precision expressed as relative standard deviation and determined individually for each analyte/concentration/matrix combination varied from 9 to 30% and was considered as acceptable. In 17 out of 28 cases, the repeatability standard deviation was the most important factor. The predominance of the repeatability compared to the other factors (matrix, days) was an indicator for the ruggedness of the assay. The validation study demonstrated that the test was able to differentiate blank samples from samples contaminated at target mycotoxin levels with a false positive rate lower than 6%. Considering realistic mycotoxin occurrence in European samples, significant economical benefits can be expected when using the test under real-world conditions.

Ca2+-dependent suicidal erythrocyte death following zearalenone exposure

Zearalenone, a cereal mycotoxin with mycoestrogen activity and effect on fertility, is known to trigger apoptosis of a variety of nucleated cell types including hematopoietic progenitor cells. In analogy to apoptosis of nucleated cells, eryptosis, the suicidal death of erythrocytes, leads to cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. The most important stimulator of eryptosis is an increase in cytosolic Ca(2+) activity ([Ca(2+)]i). The present study explored whether zearalenone triggers eryptosis. Erythrocyte volume was estimated from forward scatter, phosphatidylserine exposure at the erythrocyte surface from annexin-V binding, hemolysis from hemoglobin release, and [Ca(2+)]i from Fluo3 fluorescence. A 48-h exposure to zearalenone (≥25 μM) was followed by a significant increase in [Ca(2+)]i and annexin-V binding, and a significant decrease in forward scatter. The effect on annexin-V binding was significantly blunted in the nominal absence of extracellular Ca(2+). Zearalenone stimulates the suicidal erythrocyte death, an effect at least partially due to stimulation of Ca(2+) entry.

Mycotoxins and their effect on human health

Health risks associated with the consumption of products contaminated with mycotoxins are worldwide recognized and depend on the extent to which they are consumed in diversified diet. To some extent, the presence of small amount of mycotoxins in cereals and related food products is unavoidable; this requires risk assessments which are to be carried out by regulatory bodies in several countries to help establish regulatory guidelines for the protection of public health. By assessing the levels at which these substances in food may pose a potential risk to human health, it is possible to devise appropriate risk management strategies. However, several important factors have to be taken into account in making a rational risk management decision, such as adequate toxicological data and information concerning the extent of exposure, availability of technically sound analytical procedures (including sampling), socioeconomic factors, food intake patterns and levels of mycotoxins in food commodities which may vary considerably between countries.

Simultaneous determination of carbamate insecticides and mycotoxins in cereals by reversed phase liquid chromatography tandem mass spectrometry using a quick, easy, cheap, effective, rugged and safe extraction procedure

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 22 carbamate insecticides and 17 mycotoxins in cereals by ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Carbamates and mycotoxins were extracted from cereal samples using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) procedure without any further clean-up step. The extract was diluted with water containing 0.1% formic acid and 5.0mM ammonium acetate, and analyzed by LC-MS/MS on a Waters Acquity BEH C(18) column with water (0.1% formic acid, 0.50mM ammonium acetate)/methanol as mobile phase with gradient elution. Matrix-matched calibration was used for quantification. Blank samples (rice, wheat and corn) were fortified at 5, 10 and 50 μg/kg except for five zearalenonic compounds at 25, 50 and 250 μg/kg, and recoveries were in the range of 70-120%. Relative standard deviations were lower than 20% in all cases. The LOQ values were in the range of 0.20-29.7 μg/kg. The method is suitable for the simultaneous determination of carbamate insecticides and mycotoxins in cereals. The total time required for the analysis of one sample, including sample preparation, was about 35 min.

A novel actinomycete derived from wheat heads degrades deoxynivalenol in the grain of wheat and barley affected by Fusarium head blight

Deoxynivalenol (DON) is a hazardous and globally prevalent mycotoxin in cereals. It commonly accumulates in the grain of wheat, barley and other small grain cereals affected by Fusarium head blight (caused by several Fusarium species). The concept of reducing DON in naturally contaminated grain of wheat or barley using a DON-degrading bacterium is promising but has not been accomplished. In this study, we isolated a novel DON-utilising actinomycete, Marmoricola sp. strain MIM116, from wheat heads through a novel isolation procedure including an in situ plant enrichment step. Strain MIM116 had background degradation activity, and the activity was enhanced twofold by the consumption of DON. Among Tween 20, Triton X-100 and Tween 80, we selected Tween 80 as a spreading agent of strain MIM116 because it promoted DON degradation and the growth of strain MIM116 in the presence of DON. The inoculation of MIM116 cell suspension plus 0.01% Tween 80 into 1,000 harvested kernels of wheat and barley resulted in a DON decrease from approximately 3 mg kg(-1) to less than 1 mg kg(-1) of dry kernels, even when cells had only basal levels of DON-degrading activity. To the best of our knowledge, this is the first report that describes (1) the isolation of a DON-degrading bacterium from wheat heads, (2) the effects of surfactants on the biodegradation of DON and (3) the decrease of DON levels in naturally contaminated wheat and barley grain using a DON-degrading bacterium.

Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals

A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin–BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73–109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200μgkg−1, respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400μgkg−1, respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC–MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.

A UPLC–MS/MS for simultaneous determination of aflatoxins, ochratoxin A, zearalenone, DON, fumonisins, T-2 toxin and HT-2 toxin, in cereals

An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method is described for simultaneous determination of aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB1 and FB2), T-2 and HT-2 toxins in cereals. Mycotoxins were separated by reverse phase liquid chromatography (RP-LC) and detected by tandem mass spectrometry using an electro spray-ionization interface (ESI) in both positive- and negative- ion modes. The mean recoveries of mycotoxins from spiked cereals ranged from 83.5% to 107.3%, whereas the limit of detection (LOD) and limit of quantification (LOQ) ranged from 0.01 to 25 ng/g and 0.02–40 ng/g, respectively. The multi-mycotoxin method developed in this work was applied for the simultaneous determination of mycotoxins in 80 cereal samples collected from Malaysian markets. A total of 60 cereal samples (75%) were contaminated with at least one of these mycotoxins at levels greater than the LOD. Only one maize sample and two rice samples were contaminated at levels exceeding the European regulatory limits for aflatoxins and OTA (4 and 5 ng/g, respectively). The rates of the occurrence of mycotoxins in the commercial cereal samples were 50, 30, 19, 30, 16, 14, 14 and 12% for the aflatoxins (the total amount of AFB1, AFB2, AFG1 and AFG2), OTA, ZEA, DON, FB1, FB2, T-2 and HT-2 toxins, respectively. The results demonstrated that the procedure was suitable for the simultaneous determination of these mycotoxins in cereals and could be performed for their routine analysis in mycotoxin laboratories.

Determination of HT-2 and T-2 toxins in oats and wheat by ultra-performance liquid chromatography with photodiode array detection

European intake estimates indicate that the presence of HT-2 and T-2 toxins in cereals, mainly in oats, can be of concern for human health. Therefore, the development of sensitive, rapid and reliable methods for determining these mycotoxins in cereals, in particular oats, has high priority. A rapid ultra-performance liquid chromatographic (UPLC) method has been developed for the simultaneous determination of HT-2 and T-2 toxins in oats and wheat at μgkg−1 level. Ground samples were extracted with methanol/water (90:10, v/v) and the diluted extracts were cleaned up through immunoaffinity columns. HT-2 and T-2 toxins were separated and quantified by UPLC with photodiode array (PDA) detector (λ=202nm) in less than 5min. Mean recoveries from blank oats samples spiked with HT-2 and T-2 toxins at levels of 50–1000μgkg−1 ranged from 87 to 96%, with relative standard deviations (RSDs) lower than 7%; mean recoveries from wheat spiked with HT-2 and T-2 toxins at levels of 25–100μgkg−1 ranged from 91 to 103%, with RSDs lower than 5%. The limit of detection of the method was 8μgkg−1 for both toxins (signal-to-noise ratio 3:1). The method was successfully applied to the analysis of HT-2 and T-2 toxins in naturally contaminated oats and wheat samples. A good correlation was found by comparative analysis of naturally contaminated samples of oats (r=0.9985) and wheat (r=0.9058) using the proposed method or a reliable HPLC method with fluorescence detection after pre-column derivatization with 1-anthroylnitrile.

Determination of mycotoxins in cereals by liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC–MS/MS) method is described for simultaneous determination of aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB1 and FB2), T2 and HT2-toxin in cereals. One-step extraction using solvent mixtures of acetonitrile:water:acetic acid (79:20:1) without any clean-up was employed for extraction of these mycotoxins from cereals. The mean recoveries of mycotoxins in spiked cereals ranged from 76.8% to 108.4%. Limits of detection (LOD) and quantification (LOQ) ranged 0.01–20 and 0.02–40ng/g, respectively. The developed method has been applied for the determination of mycotoxins in 100 cereal samples collected from Malaysian markets. A total of 77 cereal samples (77%) contaminated with at least one of these mycotoxins. Occurrence of mycotoxins in commercial cereal samples were 70%, 40%, 25%, 36%, 19%, 13%, 16, and 16% for aflatoxins, OTA, ZEA, DON, FB1, FB2, T2 and HT2-toxin, respectively. The results demonstrated that the procedure was suitable for the determination of mycotoxins in cereals and could be implemented for the routine analysis.

A preliminary study of T-2 and HT-2 toxins in cereals sold in traditional market in South Korea

Fusarium species are responsible for the production of harmful trichothecenes mycotoxins in cereals. These mycotoxins are cytotoxic, potentially immunosuppressive and potent fast-acting inhibitors of protein and nucleic acid synthesis. This study validated a HPLC method for simultaneous detection of T-2 and HT-2 toxins. The method was then used for the detection of T-2 and HT-2 toxins in cereals sold in traditional markets in Gyeongnam Province, South Korea. Seventy five samples analyzed, out of which 13 and 25 samples were found to be contaminated with T-2 (35.2–431.0 ng g−1) and HT-2 (21.1–442.7 ng g−1) toxins, respectively and 4 samples were found to be contaminated with both toxins. This study provides data on the contamination level of T-2 and HT-2 toxins in cereals in traditional market in Gyeongnam province, South Korea.

Simultaneous detection of 12 mycotoxins in cereals using RP-HPLC-PDA-FLD with PHRED and a post-column derivatization system

A new method for the simultaneous quantification of 12 mycotoxins was developed and optimized using reverse phase high performance liquid chromatography (RP-HPLC) with a photodiode array (PDA) and fluorescence detector (FLD), a photochemical reactor for enhanced detection (PHRED) and post-column derivatization. The mycotoxins included aflatoxins (AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB1, FB2, and FB3), T-2 and HT-2 toxins. A double sample extraction with a phosphate-buffered saline solution (PBS) and methanol was used for co-extraction of mycotoxins, and a multifunctional immunoaffinity column was used for cleanup. Optimum conditions for separation of the mycotoxins were obtained to separate 12 mycotoxins in FLD and PDA chromatograms with a high resolution. The method gave recoveries in the range 72–111% when applied to spiked corn samples. The limits of detection (LOD) were 0.025 ng/g for AFB1 and AFG1, 0.012 ng/g for AFB2 and AFG2, 0.2 ng/g for OTA, 1.5 ng/g for ZEA, 6.2 ng/g for FB1, FB3 and HT-2 toxin, 9.4 ng/g for FB2 and T-2 toxin, and 18.7 ng/g for DON. In addition, the limits of quantification (LOQ) ranged from 0.04 ng/g for AFB2 and AFG2 to 62 ng/g for DON. The method was successfully applied to the determination of these mycotoxins in 45 cereal samples obtained from the Malaysian market. The results indicated that the method can be applied for the multi-mycotoxin determination of cereals.

Sample preparation optimization for the simultaneous determination of mycotoxins in cereals

The efficiency of three extraction solvents and three clean-up procedures was compared for simultaneous extraction and purification of aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), and zearalenone (ZEA) from spiked cereal samples. The best recovery rates for all mycotoxins were achieved using methanol: water (80:20) as the extraction solvent and AOZ multi-functional immunoaffinity column (IAC), as clean up method with recovery values of 61–89%, while that of Oasis HLB and MycoSep 226 were 37–67% and 44–78%, respectively. Then, five variables in the IAC clean-up conditions, including primary conditioning with phosphate buffer saline (PBS) (0–10 mL) (X1), extract load up volume (10–20 mL) (X2), washing volume with PBS (10–20 mL) (X3), and eluting solution volumes with methanol (1–3 mL) (X4) and acetic acid (0–1.5 mL) (X5), were optimized for the specific purification and enrichment of the mycotoxins. Results showed that primary conditioning and PBS washing did not have a significant effect on the recovery responses of mycotoxins. Optimized conditions were selected as 0, 15, 10, 1.3, and 1.5 mL for X1–X5, respectively. The recovery rates of AFB1, AFB2, AFG1, AFG2, OTA and ZEA were within 93–104% in spiked rice, under optimal conditions. LOD and LOQ were 0.0125 and 0.05 ng/g for AFB1 and AFG1, 0.0037 and 0.015 ng/g for AFB2 and AFG2, 0.05 and 0.2 ng/g for OTA, and 0.5 and 2 ng/g for ZEA, respectively. Extraction of spiked cereal samples with methanol: water (80:20) and clean up using AOZ IAC column in optimal condition provided recovery range of 77–104% for all targeted mycotoxins.

Exposure of consumers to deoxynivalenol from consumption of white bread in Hungary

In view of the frequent occurrence of mycotoxins in cereals, a study was initiated to assess the exposure of the Hungarian adult population. Consumption data for 1360 individuals, based on a 3-day questionnaire, indicated that white bread accounted for the major intake of cereal-based products. Various cereal products were analysed for 16 mycotoxins by a LC/MS/MS multi-toxin method with LOD of 16 µg kg−1 and LOQ of 50 µg kg−1. Deoxynivalenol (DON) was most frequently detected, but no acetyl-deoxynivalenol was present in detectable concentrations. Consumer exposure was calculated with standard Monte Carlo probabilistic modelling and point estimates, taking into account bread consumption and DON contamination in independently taken wheat flour and wheat grain samples. Over 55% of cases the DON intake were below 15% of the provisional maximum tolerable daily intake (PMTDI) of 1 µg/(kg bw)/day. However, in 5–15% of cases, the intake from bread consumption alone exceeded the PMTDI. Wheat grain data led to the higher percentage. Intakes estimated from both data sets were at or below the acute reference dose (ARfD) of 8 µg/(kg bw)/day in 99.94–99.97% of cases.

Analysis of multiple mycotoxins in cereals under ambient conditions using direct analysis in real time (DART) ionization coupled to high resolution mass spectrometry

Direct analysis in real time (DART) ionization coupled to an (ultra)high resolution mass spectrometer based on orbitrap technology (orbitrapMS) was used for rapid quantitative analysis of multiple mycotoxins isolated from wheat and maize by modified QuEChERS procedure. After initial evaluation of ionization efficiencies for major groups of mycotoxins achievable with DART technology, sample preparation procedure and instrument parameter settings were optimized to obtain sensitive and accurate determination of most intensively ionizing toxins (deoxynivalenol, nivalenol, zearalenon, actyldeoxynivalenol, deepoxy-deoxynivalenol, fusarenon-X, altenuene, alternariol, alternariolmethylether, diacetoxyscirpenol, sterigmatocystin). The lowest calibration levels (LCLs) estimated for the respective analytes ranged from 50 to 150 μg kg −1. Quantitative analysis was performed either with the use of matrix-matched standards or by employing commercially available 13C-labeled internal standards (available for deoxynivalenol, nivalenol and zearalenon). Good recoveries (100–108%) and repeatabilities (RSD 5.4–6.9%) were obtained at spiking level 500 μg kg −1 with isotope dilution technique. Based on matrix-matched calibration, recoveries and repeatabilities were in the range 84–118% and 7.9–12.0% (RSD), respectively. The trueness of data obtained for deoxynivalenol and zearalenon in wheat/maize by DART–orbitrapMS was demonstrated by analysis of certified reference materials (CRMs). Good agreement of these results with data generated by validated ultra-high pressure liquid chromatography–time-of-flight mass spectrometry method was documented.

Development and Comparison of Two Multiresidue Methods for the Analysis of 17 Mycotoxins in Cereals by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry

Two multiresidue methods based on different extraction procedures have been developed and compared for the liquid chromatography electrospray ionization tandem mass spectrometry analysis of 17 mycotoxins including ochratoxin A, aflatoxins (B(1), B(2), G(1), and G(2)), zearalenone, fumonisins (B(1) and B(2)), T-2 toxin, HT-2 toxin, nivalenol, deoxynivalenol, 3- and 15-acetyldeoxynivalenol, fusarenon-X, diacetoxyscirpenol, and neosolaniol in cereal-based commodities. The extraction procedures considered were a QuEChERS-like method and one using accelerated solvent extraction (ASE). Both extraction procedures gave similar performances in terms of linearity (r(2) > 0.98) and precision (both RSD(r) and RSD(iR) < 20%). Trueness was evaluated through participation in four proficiency tests and by the analysis of two certified reference materials and one quality control material. Satisfactory Z scores (|Z| < 2) and trueness values (73-130%) were obtained by the proposed procedures. Limits of quantification were similar by both methods and were within the 1.0-2.0 microg/kg range for aflatoxins, 0.5 microg/kg for ochratoxin A, and the 5-100 microg/kg range for all other mycotoxins tested. The QuEChERS-like method was found to be easier to handle and allowed a higher sample throughput as compared to the ASE method.

Development of an ELISA Reverse-Based Assay to Assess the Presence of Mycotoxins in Cereal Flour

The first successful application of the ELISA reverse method and device (ER m&d), in the context of food safety and traceability, was developed in our laboratories to detect and quantify CP4EPSPS and Cry1AB genetically modified related proteins in soy and maize samples, respectively. To prove the versatility and the transferability of the technology, the ER was here applied to assess the presence of mycotoxins in cereals. Appropriate protocols were developed to assess the presence of deoxynivalenol and ochratoxin A and the ER was tested on contaminated wheat samples. In particular, deoxynivalenol (DON) and ochratoxin A (OTA) were detected in a range of values from 430 to 5,000 µg kg−1 and from 1.7 to 10 µg kg−1, respectively. The assays were optimized to reach a limit of quantification equal to 550 and 1.8 µg kg−1 for DON and OTA, respectively.

Solid-phase extraction using molecularly imprinted polymers for selective extraction of a mycotoxin in cereals

The aim of this work was to develop a method for the clean-up of a mycotoxin, i.e. Ochratoxin A (OTA), from cereal extracts employing a new molecularly imprinted polymer (MIP) as selective sorbent for solid-phase extraction (SPE) and to compare with an immunoaffinity column. A first series of experiments was carried out in pure solvents to estimate the potential of the imprinted sorbent in terms of selectivity studying the retention of OTA on the MIP and on a non-imprinted polymer using conventional crushed monolith. The selectivity of the MIP was also checked by its application to wheat extracts. Then, after this feasibility study, two different formats of MIP: crushed monolith and micro-beads were evaluated and compared. Therefore an optimization procedure was applied to the selective extraction from wheat using the MIP beads. The whole procedure was validated by applying it to wheat extract spiked by OTA at different concentration levels and then to a certified contaminated wheat sample. Recoveries close to 100% were obtained. The high selectivity brought by the MIP was compared to the selectivity by an immunoaffinity cartridge for the clean-up of the same wheat sample. The study of capacity of both showed a significant higher capacity of the MIP.