Novel analytical methods for Fusarium toxins in the cereal food chain

Transcription

1 Novel analytical methods for Fusarium toxins in the cereal food chain Nouvelles méthodes pour l analyse des mycotoxines de Fusarium dans la chaîne de transformation des céréales Visconti, Angelo ; De Girolamo, Annalisa ; Lattanzio, Veronica M. T. ; Lippolis, Vincenzo ; Pascale, Michelangelo ; Solfrizzo, Michele Institute of Sciences of Food Production, National Research Council (ISPA-CNR), Via G. Amendola 122/O, 70126, Bari, Italy angelo.visconti@ispa.cnr.it Abstract An overview of novel analytical methods recently developed at ISPA-CNR for the determination of the major Fusarium toxins occurring in cereals and cereal-based products is presented. These methods deal in particular with: (i) fluorescence polarization (FP) immunoassay for rapid quantification of deoxynivalenol (DON) in common wheat, durum wheat, semolina and pasta; (ii) Fourier transform-near infrared spectroscopy (FT-NIR) for rapid, non-destructive and quantitative determination of DON in wheat; (iii) HPLC with fluorescence detection for simultaneous determination of T-2 toxin (T- 2) and HT-2 toxin (HT-2) in cereal grains at ppb levels using immunoaffinity column clean-up, and specific labeling reagents; (iv) liquid chromatographytandem mass spectrometry (LC-MS/MS) for simultaneous determination of DON, nivalenol (NIV), T-2 and HT-2 in cereals and cereal-based food products after extract clean-up by Oasis HLB columns and (v) LC-MS/MS for simultaneous determination of DON, T-2, HT-2, zearalenone (ZEA) and fumonisins (FBs), together with aflatoxins (AFs) and ochratoxin A (OTA), in maize after clean-up by a multimycotoxin immunoaffinity column. Basic principles, performances, advantages and limitations of these methods are reviewed. Keywords: Fusarium toxins, fluorescence polarization immunoassay, FT- NIR spectroscopy, LC-MS/MS, multimycotoxin immunoaffinity columns. 1

2 Résumé Un panorama des nouvelles méthodes d analyse développées récemment par le laboratoire ISPA-CNR pour la quantification des mycotoxines de Fusarium ayant la plus forte occurrence dans les matrices céréales ou les produits dérivés est présenté. Ces méthodes sont basées sur différentes technologies et en particulier: (i) le test immuno-enzymatique à polarisation de fluorescence (FP) pour la quantification rapide du déoxynivalénol (DON) dans le blé tendre, le blé dur, les semoules et les pâtes; (ii) la spectroscopie dans le proche infrarouge à transformée de Fourier (FT-NIR) pour la détermination rapide non-destructive des teneurs en DON dans le blé; (iii) la CLHP avec détecteur fluorimétrique pour la détection simultanée de les toxines T2 (T-2) et HT-2 (HT-2) dans les grains de céréales à des teneurs de l ordre du ppb, utilisant des colonnes d immuno-affinité pour la purification et des réactifs spécifiques de marquage ; (iv) la chromatographie liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS) pour la détection simultanée du DON, du nivalénol (NIV), T-2 et HT-2 dans les céréales et les aliments à base de céréales en utilisant des colonnes d extraction / purification Oasis HLB ; (v) LC-MS/MS pour la détermination simultanée de DON, T-2, HT-2, la zéaralénone (ZEA), et les fumonisines (FBs), ensemble avec les aflatoxines (AFs) et l ochratoxine A (OTA), dans le maïs après purification sur colonne d immuno-affinité multitoxines. Les principes de base, les performances, les avantages et les inconvénients de ces méthodes sont passés en revue. Mots-clés : Toxines de Fusarium, immunotest polarisation fluorescence, spectroscopie FT-NIR, LC-MS/MS, colonne immuno-affinité multitoxines Introduction Fusarium species are plant pathogens commonly associated with cereals that, under favourable environmental conditions, can produce several secondary toxic metabolites. The major Fusarium toxins found in cereals and cereal-based products that can be harmful to both human and animal health are some trichothecenes, such as deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZEA) and fumonisins (FB 1, FB 2 and FB 3 ) (Visconti, 2001). The Scientific Committee on Food (SCF) of the European Commission has recently given several opinions on Fusarium toxins evaluating DON, ZEA, FB 1, FB 2 and FB 3, NIV, T-2 and HT-2, based on the toxicological information available (European Commission, 2006a). In these evaluations the Committee established a full Tolerable Daily Intake (TDI) 2

3 for DON (1 μg/kg body weight/day) and for FB 1, FB 2 and FB 3, alone or in combination (2 μg/kg b.w./day), and a temporary TDI (t-tdi) for NIV (0.7 μg/kg b.w./day) and ZEA (0.2 μg/kg b.w./day) and a combined t-tdi for T-2 and HT-2 (0.06 μg/kg b.w./day). A recent SCOOP project, aiming to evaluate the risk of dietary exposure to Fusarium toxins by the population of EU member states, showed that Fusarium toxins are widely distributed in the food chain in the EU and the major sources of dietary intake of Fusarium toxins are cereal products, mainly based on wheat and maize. The dietary intakes for all populations and adults were generally below the TDIs for the respective toxins, whereas for risk groups like infants and young children they were close to or exceeded the TDI in some cases (European Commission, 2003). In order to protect human health from exposure to these mycotoxins through the consumption of cereal-based food products the European Commission has recently established regulatory limits for DON, ZEA and fumonisins (sum of FB 1 and FB 2 ) in raw materials and products intended for human consumption, while permissible levels of T-2 and HT-2 in cereal-based products are under discussion (European Commission, 2005; 2006a). The Commission has also issued recommendations to prevent or reduce the contamination of Fusarium toxins in cereals and cereal products (European Commission, 2006b). There is a need to develop and validate analytical methods for rapid, sensitive and accurate determination of these mycotoxins in cereals and cereal-based products in order to properly assess the relevant risk of exposure and to ensure that regulatory levels fixed by the EU or other international organizations are met. Advances in the analysis of Fusarium mycotoxins in cereals and its quality assurance have been reviewed by several authors (Krska et al., 2001; Pascale & Visconti, 2007; van Osenbruggen & Petterson, 2005). A variety of emerging methods have been reported for the rapid analysis of mycotoxins (including Fusarium toxins). They are based on novel technologies including lateral flow devices (LFD), membrane-based flow-through enzyme immunoassay, fluorescence polarization (FP) immunoassay, near-infrared spectroscopy (NIR), molecularly imprinted polymers (MIP) and surface plasmon resonance (SPR) biosensors (Goryacheva et al., 2007; Krska et al., 2005; Maragos 2004; Pascale & Visconti, 2007; Zheng et al., 2006). This paper summarizes recent results obtained in our laboratory in the area of analytical chemistry of Fusarium mycotoxins applied to cereals and cereal-based products. In particular, novel methods have been developed based on: fluorescence polarization (FP) and Fourier transform-near infrared spectroscopy (FT-NIR) for rapid quantification of DON in wheat and derived products; HPLC with fluorescence detection for simultaneous determination of T-2 and HT-2 in cereal grains; liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneous determination of DON, NIV, T-2 and HT-2 in cereals and cereal-based food products after Oasis HLB columns clean-up, and for simultaneous determination of DON, T-2, HT-2, ZEA and FBs, together with aflatoxins (AFs) and ochratoxin A (OTA), in maize after multimycotoxin immunoaffinity column clean-up. 3

4 1. Fluorescence polarization immunoassay for rapid quantification of deoxynivalenol in wheat and derived products Fluorescence polarization (FP) immunoassay is a rapid technique measuring interactions between a fluorescently labelled antigen (tracer) and a specific antibody, and has been extensively used in human and veterinary diagnostics. Only recently FP immunoassays have been reported for the analysis of mycotoxins, including AFs, FBs, ZEA, OTA and DON, although their application to cereal samples resulted in poor accuracy and sensitivity (Maragos & Plattner, 2002; Maragos & Kim, 2004; Nasir & Jolley, 2003; Shim et al., 2004). In particular, the FP immunoassay developed for screening DON in wheat kernels, when compared with an HPLC/UV method, showed an overestimation of DON in naturally contaminated samples which did not allow accurate measurements of the toxin at levels close to the EU regulatory limits (Maragos & Plattner, 2002). We have developed an FP immunoassay for the determination of DON in common wheat, durum wheat, semolina and pasta (Lippolis et al., 2006). The FP immunoassay was based on the competition for a DON specific monoclonal antibody between DON and a DONfluorescent tracer obtained by selective reaction of DON (at the C-3 hydroxyl position) with 4 -(aminomethyl)fluorescein. The analytical method consisted of a rapid extraction of DON with phosphate buffered saline (PBS), followed by filtration through paper and glass microfibre filters and FP immunoassay quantification. The overall time for DON analysis in wheat based products, including sample preparation and FP immunoassay measurement, was about 10 minutes (Lippolis et al., 2006). By comparison with a validated HPLC/immunoaffinity clean-up method (Mac Donald et al., 2005), a consistent overestimation of DON content was observed in both spiked and naturally contaminated samples of durum wheat, common wheat, semolina and pasta (free of 3-acetyl-DON and 15-acetyl-DON). The FP background signal was accurately measured (FP analysis of 150 samples at different DON spiking levels and different amounts of analyzed matrix) and shown to be directly proportional to the amount of analyzed matrix independent of the real DON content in the sample. These results showed that DON overestimation was due to a matrix effect and could not be attributed to the presence of other fungal metabolites that cross-react with the DON antibody, as previously hypothesized (Maragos & Plattner, 2002). After subtracting the DON background level for common wheat (0.39 µg/g DON), durum wheat (0.27 µg/g DON), semolina (0.08 µg/g DON) and pasta (0.04 µg/g DON), average recoveries (from samples spiked at levels ranging from 0.25 to 1.75 µg/g) were 100%, 98%, 102% and 101%, respectively, with relative standard deviations (RSD) lower than 5% (n = 4). The limit of detection (calculated as three standard deviations of the FP signal of the blank sample, n = 10) was 0.08 µg/g for all matrices, and the applicability range of the assay was from 0.08 to 2.00 µg/g DON. Comparative analyses of naturally contaminated samples of durum wheat, common wheat, semolina and pasta showed a good correlation (r > ) between DON concentrations 4

5 obtained by the FP immunoassay (after subtracting the DON background level) or the HPLC/immunoaffinity clean-up method. The optimized FP method for the determination of DON in wheat and derived products showed better sensitivity and accuracy with respect to the previously reported FP immunoassay and better accuracy and precision with respect to the HPLC/immunoaffinity clean-up method. In addition, a fully automated FP prototype (European patent pending) has been developed in our laboratory by assembling an FP reader with an autosampler assisted by a PC through a specific software for data handling (Figure 1). The automated FP immunoassay system offers a rapid, high-throughput, reliable and easy-to-use tool for monitoring DON in wheatbased products in order to fulfill regulatory levels and protect consumer health from the risk of exposure to the toxin. The assay is easy to perform, and is quite useful for DON screening at levels that occur naturally in wheat and wheat based products. Figure 1. The automated FP prototype for the determination of DON. 2. Fourier transform-near infrared spectroscopy for rapid determination of deoxynivalenol in wheat In the past few decades, an increasing use of infrared spectroscopy as a rapid and nondestructive technology is being recorded for the analysis of different compounds in a broad 5

6 range of foods. Recently, the use of near-infrared (NIR) and mid-infrared (MIR) spectroscopy has been evaluated also for rapid determination of mycotoxins in cereals. NIR transmittance was used for the determination of DON in wheat kernels at levels above 500 µg/kg (Petterson & Aberg, 2003). Other authors reported the use of NIR reflectance measurements for prediction of scab, DON and ergosterol content in single kernels of highly infected wheat (Delwiche & Hareland, 2004; Dowell et al., 1999). NIR reflectance spectroscopy was used also to monitor mold contamination in post-harvest maize and to segregate FB 1 contaminated maize from uncontaminated maize (Berardo et al., 2005). A method for the detection of Fusarium contamination on maize using Fourier transform (FT)-MIR spectroscopy with attenuated total reflection (ATR) was also described. The method enabled the segregation of DON contaminated maize from uncontaminated one (Kos et al., 2007). FT-MIR-ATR was also used for the determination of aflatoxins in groundnut and groundnut cakes (Mirghani et al., 2001). A rapid method using FT-NIR has been developed in our laboratory for the quantitative determination of DON in durum and common wheat. Partial Least Square (PLS) regression models were built in the spectral range cm -1 by using 16 and 31 ground calibration samples of durum wheat (cv. Duilio) and common wheat (cv. Serio), respectively, with particle size < 500 µm (Cyclotec mill, Tecator, Sweden). Mean DON levels ranged from 188 to 2100 µg/kg in durum wheat and from not detected (< 80 µg/kg) to 1741 µg/kg in common wheat. DON reference measurements were performed by HPLC (Mac Donald et al., 2005). Correlation coefficients between reference DON measurements and FT-NIR predicted DON levels, root mean square error of calibration (RMSEC), root mean square error of cross-validation (RMSECV) and bias showed a good quality of the regression models. The predicted residual error sum of squares (PRESS) value was used as a good indicator of the measurement uncertainty of the model to predict blind samples. In the development of the calibration models, 10 partial least squares (PLS) factors were set up as a maximum number to work with. FT-NIR spectra were recorded using an Antaris II FT-NIR spectrophotometer (Thermo Electron Corporation, USA) equipped with an interferometer, an integrating sphere working in diffuse reflection, and an indium and gallium arsenide (InGaAs) detector. PLS regressions had correlation coefficients (r) of for both durum and common wheat, whereas RMSEC and RMSECV were 55 µg/kg and 350 µg/kg DON in durum wheat, and 50 µg/g and 319 µg/kg in common wheat, respectively. PLS regression models were validated in the range of 250-1,160 µg/kg for durum wheat and 0-1,260 µg/kg for common wheat. The ability of the FT-NIR to qualitatively discriminate between blank (< 80 µg/kg DON) and naturally contaminated durum wheat and common wheat samples (ground at the same particle sizes, < 500 µm) belonging to different cultivars was evaluated by discriminant analysis. A total of 48 durum wheat and 47 common wheat samples were analyzed for DON by the HPLC reference method, and samples were split in two classes, one relevant to blank samples and the other one relevant to naturally contaminated samples. Two additional classes were obtained by spiking some blank wheat samples (durum and common wheat) with DON standard solutions at levels ranging from 200 to µg/kg. Wheat samples were classified as members of each group based on the lowest 6

7 corresponding Mahalanobis distance, i.e. the multi-dimensional space whose boundaries determine the variation range. Discriminant analysis correctly discriminated durum wheat from common wheat samples as well as contaminated from blank samples. The two classes of spiked samples were also well identified. Only 4.2% of both durum and common contaminated wheat samples were misclassified as blank samples. Figure 2 shows the score/score plot of PC1 and PC2 resulting from the discriminant analysis, indicating that PC1 and PC2 accounted for 69.0% and 26.5%, respectively, of the total variance. Common wheat Durum wheat Naturally contaminated Naturally contaminated Spiked Blank Spiked Blank Figure 2. Score/score plot of spectral data (PC1 vs PC2) of 48 durum wheat samples and 47 common wheat samples belonging to different varieties. The two clusters represent common (left) and durum (right) wheat both containing blank ( 80 µg/kg DON), spiked and naturally contaminated samples. 3. HPLC with fluorescence detection for simultaneous determination of T-2 and HT-2 toxins in cereal grains Gas-chromatography (GC) with electron-capture (ECD) or mass spectrometric (MS) detection is largely used for quantification of type-a trichothecenes (including T-2 and HT- 2). A comparative collaborative study supported by the EU showed poor method performances in GC analysis of trichothecenes in terms of recovery, accuracy, and precision. The main problems were attributed to matrix interferences giving rise to trichothecene response enhancement (Petterson & Langseth, 2002). Liquid chromatography 7

8 coupled with mass spectrometry (LC/MS) has been recently applied to the simultaneous determination of major type-a and type-b trichothecenes as well as zearalenone, but problems of accuracy due to matrix effects were observed (Berthiller et al., 2005). HPLC with fluorescence detection (FD) provides high sensitivity, selectivity and repeatability of measurements, but it is not applicable to the detection of trichothecenes lacking of fluorophore groups in their chemical structure. The simultaneous determination of T-2, HT-2, T-2 triol and T-2 tetraol by HPLC/FD was reported using pre-column derivatization with coumarin-3-carbonyl chloride as fluorescence label (Cohen & Boutin- Muma, 1992). This derivatizing reaction was used for developing a method for the determination of T-2, HT-2, neosolaniol and diacetoxyscirpenol in cereal cultures of Fusarium sporotrichioides on maize, rice and wheat by HPLC/FD after solid phase extraction (SPE) column clean-up. Although the method had good sensitivity with standard toxins, when applied to cereal samples it showed poor toxin recoveries (Mateo et al., 2002). Recently we have shown 1-anthroylnitrile (1-AN) to be an efficient fluorescent labelling reagent for T-2 and HT-2 under mild conditions (Visconti et al., 2005). The derivatizing reaction was used to develop a sensitive, reproducible and accurate method for the simultaneous determination of T-2 and HT-2 in wheat, maize and barley by HPLC/FD after immunoaffinity column clean-up. Recoveries from spiked samples with toxin levels from 25 to 500 µg/kg ranged from 70 to 100%, with relative standard deviations (RSD) lower than 8%. The method allowed the determination of T-2 and HT-2 at levels of 5 µg/kg and 3 µg/kg (based on a signal-to-noise ratio of 3), respectively. However, the method did not allow the determination of HT-2 in oats because of interfering chromatographic peaks occurring at the retention time of the HT-2-(1-AN) derivative (Visconti et al., 2005). COCN COCN 1-anthroyl cyanide (1-anthroylnitrile, 1-AN) COCl pyrene 1-carbonyl cyanide (PCC) COCl 1-naphthoyl chloride (1-NC) 2-naphthoyl chloride (2-NC) Figure 3. Fluorescence labeling reagents for T-2 and HT-2 toxins 8

9 In order to improve the detection limit of the HPLC/FD method for T-2 and HT-2 in foodstuffs, including oats, three different commercially available fluorescent reagents were tested as labelling dyes (Figure 3). 1-naphthoyl chloride (1-NC), 2-naphthoyl chloride (2- NC) and pyrene-1-carbonyl cyanide (PCC) reacted with the hydroxyl groups of T-2 and HT-2 under mild conditions to form the corresponding esters (Lippolis et al., 2007). A wide linear range ( ng for either T-2 or HT-2 derivatized toxin), good repeatability (RSD 8%) of the reaction, and good stability (up to 2 weeks at -20 C and five days at room temperature) of the fluorescent derivatives were observed. Detection limits were 10.0, 6.3 and 2.0 ng for derivatized T-2, and 6.3, 2.3 and 2.8 ng for derivatized HT-2 with 1-NC, 2- NC and PCC, respectively. A higher fluorescent intensity was observed for derivatives obtained by reaction of T-2 and HT-2 with PCC as compared to those obtained with 1-AN. Preliminary studies showed the applicability of the new labelling reagents (PCC or 2-NC) for the simultaneous determination of T-2 and HT-2 by HPLC/FD after immunoaffinity column clean up in naturally contaminated cereal grains, including oats (Lippolis et al., 2007). 4. Liquid chromatography-tandem mass spectrometry for simultaneous determination of Fusarium toxins Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is spreading rapidly into the field of mycotoxin analysis. Besides the high sensitivity, tandem mass spectrometry provides also the highest degree of certainty in analyte identification and may be employed in accordance with recent European Union guidelines to obtain data with relevant unambiguity (European Commission, 2002). Moreover, the high selectivity of LC-MS/MS instruments enables to reduce or even omit sample preparation, increasing sample throughput. An overview on the application of LC-MS in the analysis of frequently occurring mycotoxins, including trichothecenes, FBs and ZEA, has been recently published (Zöllner & Mayer-Helm, 2006) LC-MS/MS for simultaneous determination of nivalenol, deoxynivalenol, T-2 and HT-2 toxins in cereals and cerealbased products LC-MS/MS is becoming the technique of choice for the simultaneous determination of type-a and type-b trichothecenes (Berger et al., 1999; Berthiller et al., 2005; Biselli & Hummert, 2005; Cavaliere et al., 2005) overcoming traditional drawbacks of gas chromatographic methods with respect to recovery, accuracy, and precision of the measurements (Petterson & Langseth, 2002). Extract clean-up for the determination of trichothecenes in cereals is frequently done by MycoSep #227 columns (Romer Labs, USA), which have also been successfully applied in several LC-MS methods, although low NIV recoveries have been reported (Berthiller, et al. 2005, Biselli & Hummert, 2005). 9

10 An LC-APCI-MS/MS method for the simultaneous determination of NIV, DON, T-2 and HT-2 in cereals and cereal-based foods such as wheat, maize, barley, infant foods, cereal snacks and biscuits has been recently developed in our laboratory (Lattanzio et al. 2007a). A clean up procedure, based on reversed phase SPE Oasis HLB columns (Waters, USA) was used, allowing good recoveries for all studied trichothecenes that vary considerably in polarity. In particular, recovery of NIV (the most polar trichothecene) resulted significantly improved as compared with those obtained by using MycoSep #227 columns for extract clean up. In order to assess its general applicability and robustness among a wide range of cereals and cereal-based foods, the whole analytical procedure was validated in three different cereals (wheat, barley and maize) and five cereal based foods (infant semolina, infant biscuits, bacon biscuits, cocoa wafers and coconut snacks). Mean detection limits, based on a signal-to-noise ratio of 3, in the various investigated matrices, were 3.0 µg/kg for NIV, 4.2 µg/kg for DON, 0.8 µg/kg for HT-2, and 0.5 µg/g for T-2. Mean recovery values, obtained from cereals and cereal products spiked with NIV, DON, T-2 and HT-2 at spiking levels from 10 to 1000 µg/kg, ranged from 72 to 110% with mean RSD lower than 10%. A new approach for matrix effect evaluation has been developed throughout this study. Matrix effects in LC-MS analysis are well known and various approaches have been proposed to overcome this problem (Biselli & Hummert, 2005; Häubl et al., 2006; Razzazi- Fazeli et al., 2002; Sulyok et al., 2006). However a detailed investigation has never been carried out. A systematic study of matrix effects in different cereals and cereal products was carried out by statistically comparing (Student t test) the slopes of standard calibration curve with matrix-matched calibration curve for each of the toxins and the matrices considered for method validation. Statistically significant matrix effects were observed for seven out of the eight matrices tested, indicating that for accurate quantitative analysis a matrix-matched calibration is necessary. Moreover, the slope of the regression varied considerably from matrix to matrix also revealing changing influences of co-eluting matrix compounds on the analyte signal. On the basis of these findings we concluded that, due to the observed matrix effects, the matrix-matched calibration is necessary for reliable quantitative analyses LC-MS/MS for simultaneous determination of Fusarium toxins, aflatoxins and ochratoxin A in maize There is currently a strong trend toward multi-mycotoxin methods for the simultaneous determination of mycotoxins belonging to different chemical families. This topic has been recently investigated by several authors, although major problems related to the extraction and clean up steps have not been adequately undertaken and solved (Cavaliere et al., 2005; Ren et al., 2007; Sulyok et al., 2007; Tanaka et al., 2006). An LC-ESI-MS/MS multiresidual method was developed for the simultaneous determination of all main Fusarium toxins (DON, T-2, HT-2, FB 1, FB 2, ZEA) together with aflatoxins (AFBB1, AFB 2, AFG 1, AFG 2 ) and OTA in maize (Lattanzio et al., 2007b). A double extraction, using PBS and methanol/water, was performed to achieve effective coextraction of all mycotoxins under investigation. For extract clean up a new multitoxin immunoaffinity column (AOFZDT2, Vicam, USA) containing antibodies for all these 10

11 mycotoxins was successfully used. A typical chromatogram relevant to a maize sample spiked with the 11 mycotoxins is reported in Figure 4. Method performances, i.e. recovery, repeatability and linearity over the working range, were evaluated for each toxin at contamination levels comparable with the relevant established or expected EU maximum limits in maize (European Commission, 2006a). Recovery values for the whole analytical procedure ranged from 79% to 104% with RSD lower than 13% for all tested toxins. Limits of detection (LOD, signal-to-noise ratio of 3) were: 4.2 µg/kg for DON, 1.1 for µg/kg FBB1, 0.4 µg/kg for FB 2, 1.9 µg/kg for HT-2, 1.5 µg/kg for T-2, 0.7 µg/kg for ZEA, 0.8 µg/kg for AFG 2, 0.4 µg/kg for AFG 1, 0.3 µg/kg for AFB 2, 0.6 µg/kg for AFB 1B, and 0.6 µg/kg for OTA. Based on the obtained LOD values, the method allows to assess, in a single analysis, the compliance of maize with the EU maximum permitted levels for OTA, DON, and ZEA, while providing quantitative data on AFs contamination at levels slightly above the EU regulated levels. It is also quite satisfactory for levels of FBs, T-2 and HT-2 which levels will soon be regulated or are currently under discussion within the EU. 100 ZEA (-) (+) (+) (-) (+) x 3.0 DON FB 1 % T-2 FB 2 AFG 1 AFB 1 AFB 2 HT-2 OTA AFG Time, min Figure 4. Total ion chromatogram (TIC of MRM) of an extract of maize spiked at levels of 500 µg/kg DON; 2 µg/kg AFG 2, AFBB2; 6 µg/kg AFG 1 ; 10 µg/kg AFB 1 ; 500 µg/kg FB 1 ; 250 µg/kg FB 2 ; 100 µg/kg T-2, HT-2, ZEA; 20 µg/kg OTA; Vertical lines indicate the periods of polarity switching. HPLC conditions: column: Gemini RP18 ( mm, 5 µm); mobile phase: methanol/water containing 0.5% acetic acid, 1 mm ammonium acetate, flow rate 200 µl/min, without splitting (from Lattanzio et al., 2007, Rapid Communications in Mass Spectrometry, 21, p ). 11

12 Conclusions The novel analytical methods presented herein meet the need of quality control laboratories for reliable and rapid (automated) methods for (simultaneous) determination of major Fusarium toxins occurring in cereals and cereal-based products (Table 1). Table 1. Advantages and disadvantages of novel methods for Fusarium toxins. Method Fusarium toxins Clean-up Advantages Disadvantages FPIA DON no Rapid No clean up Automated FT-NIR DON no Rapid No extraction No clean up Easy-to-use HPLC/FD T-2, HT-2 IAC Good sensitivity Good selectivity Good repeatability Antibody crossreactivity Specific calibration models needed Expensive equipment Calibration model validation needed Statistical basis required Expensive equipment Derivatization required HPLC/MS- MS DON, NIV, T-2, HT-2 SPE column (Oasis HLB) Multiple mycotoxins analysis Good sensitivity Analyte confirmation No derivatization Very expensive equipment Expert personnel Matrix assisted calibration curve HPLC/MS- MS DON, T-2, HT-2, ZEA, FB 1, FBB2 Multimycotoxin IAC (AOFZDT2 ) (see above) (see above) FPIA = fluorescence polarization immunoassay; FT-NIR = Fourier transform-near infrared spectroscopy; HPLC/FD = high-performance liquid chromatography/fluorescence detector; HPLC/MS-MS = high-performance liquid chromatography-tandem mass spectroscopy; IAC = immunoaffinity column; SPE = solid phase extraction. In particular, the fully automated FP immunoassay allows rapid and quantitative determination of DON at levels that can naturally occur in wheat and wheat based products, and can be used as an alternative method to HPLC/immunoaffinity clean-up. The overall time for DON analysis in wheat based products, including sample preparation and FP 12

13 immunoassay measurement, was about 10 minutes, thus allowing a much higher throughput of analyses as compared to HPLC or GC methods. FT-NIR instruments are faster and more sensitive than traditional dispersive NIR instruments and allow simultaneous measurements of full spectra with higher resolution. FT-NIR spectroscopy is a promising technique for rapid (less than 10 min) and quantitative determination of DON at levels below the maximum limits set by the European Union for unprocessed durum and common wheat. New fluorescent labeling reagents (1-AN, 1-NC, 2-NC and PCC) have been shown to react with T-2 and HT-2 under mild conditions to give the corresponding fluorescent esters. These reagents have been used to develop and validate a sensitive and accurate analytical method for the simultaneous determination of T-2 and HT-2 in raw cereals, including oats, by HPLC/FD after immunoaffinity column clean-up. The potential of LC-MS/MS for simultaneous and high sensitive detection of several Fusarium toxins (also in combination with AFs and OTA) in cereals and cereal-based products has also been shown. Different clean up strategies, based on reversed phase SPE cartridges (Oasis HLB) or multitoxins immunoaffinity columns (AOFZDT2 ) have been developed for one-step purification of several mycotoxins. Performance characteristics of the developed methods indicate that LC-MS/MS can lead to fully validated analytical methods according to EU requirements for certified mycotoxin analysis. References Berardo N., Pisacane V., Battilani P., Scandolara A., Pietri A., Marocco A., Rapid detection of kernel rots and mycotoxins in maize by near-infrared reflectance spectroscopy. Journal of Agricultural and Food Chemistry, 53, p Berger U., Oehme M., Kuhn F., Quantitative determination and structure elucidation of type-a and B-trichothecenes by HPLC/ion trap multiple mass spectrometry. Journal of Agricultural and Food Chemistry, 47, p Berthiller F., Schuhmacher R., Buttinger G., Krska R., Rapid simultaneous determination of major type A- and B-trichothecenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography A, 1062, p Biselli S., Hummert C., Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples. Food Additives and Contaminants, 22, p Cavaliere C., Foglia P., Pastorini E., Saperi R., Laganà A., Development of a multiresidue method for analysis of major Fusarium mycotoxins in corn meal using liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 19, p Cohen H., Boutin-Muma B., Fluorescence detection of trichothecene mycotoxins as coumarin- 3-carbonyl chloride derivatives by high-performance liquid chromatography. Journal of Chromatography, 595, p Delwiche S.R., Hareland G.A., Detection of scab-damaged hard red spring wheat kernels by near-infrared reflectance. Cereal Chemistry, 81, p Dowell F.E., Ram M.S., Seitz M.S., Predicting scab, vomitoxin, and ergosterol in single wheat kernels using near-infrared spectroscopy. Cereal Chemistry, 76, p

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