Novel analytical methods for Fusarium toxins in the ... - SympoScience

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
e-mail: 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.
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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
(FB1, FB2 and FB3) (Visconti, 2001).
The Scientific Committee on Food (SCF) of the European Commission has recently given
several opinions on Fusarium toxins evaluating DON, ZEA, FB1, FB2 and FB3, 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)
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for DON (1 μg/kg body weight/day) and for FB1, FB2 and FB3, 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 FB1 and FB2) 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.
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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 > 0.9955) between DON concentrations
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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
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ange 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 FB1 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 10000 - 4000
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 0.996 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 10000 µg/kg.
Wheat samples were classified as members of each group based on the lowest
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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
Naturally
contaminated
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
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Naturally
contaminated
Spiked
Durum wheat
Blank

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).
1-anthroyl cyanide
(1-anthroylnitrile, 1-AN)
COCl
COCN
1-naphthoyl chloride
(1-NC)
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COCN
pyrene 1-carbonyl cyanide
(PCC)
COCl
2-naphthoyl chloride
(2-NC)
Figure 3. Fluorescence labeling reagents for T-2 and HT-2 toxins

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 (10-1000 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).
4.1. 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).
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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.
4.2. 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, FB1, FB2, ZEA) together with
aflatoxins (AFBB1, AFB2, AFG1, AFG2) 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
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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 FB2, 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 AFG2, 0.4 µg/kg for AFG1, 0.3 µg/kg for
AFB2, 0.6 µg/kg for AFB1B , 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
%
0
ZEA
(-) (+) (+) (-) (+)
DON
AFG 2
x 3.0
AFG 1
AFB 2
AFB 1
HT-2
FB 1
5 10 15 20 25 30 35 40 45 50 55
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 AFG2, AFBB2; 6 µg/kg AFG1; 10 µg/kg AFB1; 500 µg/kg FB1; 250 µg/kg FB2; 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
(150 × 2.0 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. 3253-3261).
T-2
FB 2
OTA
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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
HPLC/MS-
MS
HPLC/MS-
MS
DON, NIV,
T-2, HT-2
DON, T-2,
HT-2, ZEA,
FB1, FBB2
SPE column
(Oasis ® HLB)
Multimycotoxin
IAC (AOFZDT2 ® )
Colloque Fusariotoxines des Céréales – Arcachon - 11–13 septembre 2007
www.symposcience.org
12
Multiple mycotoxins
analysis
Good sensitivity
Analyte confirmation
No derivatization
Antibody crossreactivity
Specific calibration
models needed
Expensive
equipment
Calibration model
validation needed
Statistical basis
required
Expensive
equipment
Derivatization
required
Very expensive
equipment
Expert personnel
Matrix assisted
calibration curve
(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

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.
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Colloque Fusariotoxines des Céréales – Arcachon - 11–13 septembre 2007
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