Zbornik Matice srpske za prirodne nauke / Proc. Nat.

Sci, Matica Srpska Novi Sad, 117, 1525, 2009

UDC 633.1:615.918.099 DOI:10.2298/ZMSPN0917015P

M i c h e l a n g e l o N. P a s c a l e
Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Via G. Amendola 122/O, 70126 Bari, Italy

DETECTION METHODS FOR MYCOTOXINS IN CEREAL GRAINS AND CEREAL PRODUCTS

ABSTRACT: Analytical methods for mycotoxins in cereals and cereal-based products require three major steps, including extraction, clean-up (to eliminate interferences from the extract and concentrate the analyte), and detection/determination of the toxin (by using suitable analytical instruments/technologies). Clean-up is essential for the analysis of mycotoxins at trace levels, and involves the use of solid phase extraction and multifunctional (e.g. MycoSep) or immunoaffinity columns. Different chromatographic methods are commonly used for quantitative determination of mycotoxins, including gas-chromatography (GC) coupled with electron capture, flame ionization or mass spectrometry (MS) detectors (mainly for type-A trichothecenes), and high-performance liquid chromatography (HPLC) coupled with ultraviolet, diode array, fluorescence or MS detectors. The choice of method depends on the matrix and the mycotoxin to be analyzed. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is spreading rapidly as a promising technique for simultaneous screening, identification and quantitative determination of a large number of mycotoxins. In addition, commercial immunometric assays, such as enzyme-linked immunosorbent assays (ELISA), are frequently used for screening purposes as well. Recently, a variety of emerging methods have been proposed for the analysis of mycotoxins in cereals based on novel technologies, including immunochromatography (i.e. lateral flow devices), fluorescence polarization immunoassays (FPIA), infrared spectroscopy (FT-NIR), molecularly imprinted polymers (MIPs) and optical biosensors. KEYWORDS: cereals, mycotoxins, rapid methods, clean-up, GC, HPLC, immunoassays, LC-MS/MS, molecularly imprinted polymers

INTRODUCTION Major mycotoxins that can occur in cereal grains and cereal-based products are Fusarium mycotoxins, deoxynivalenol (occurring mainly in wheat, maize, barley, oats, rye), T-2 and HT-2 toxins (oats, wheat, barley), zearalenone (maize, wheat) and fumonisins (maize), and Aspergillus or Penicillium mycotoxins, aflatoxins (maize) and ochratoxin A (maize, wheat, barley, rye). Human or animal exposure to these natural contaminants can lead to acute or chronic diseases, and in some cases death (R i c h a r d, 2007). According to 15

the results of risk assessment studies (SCOOP projects), cereals and cereal-based products are the main source of mycotoxin intakes by the EU population (http://ec.europa.eu/food/food/chemicalsafety/contamin ants/index_en.htm). In order to protect human health from exposure to these mycotoxins through the consumption of cereal-based foods, the European Commission has recently established regulatory limits for deoxynivalenol (DON), zearalenone (ZEA), fumonisins (sum of FB1 and FB2), aflatoxins (AFB1 and sum of AFB1, AFB2, AFG1 and AFG2) and ochratoxin A (OTA) in raw cereals and derived products intended for human consumption, while permissible levels of T-2 and HT-2 toxins in cereals are under discussion (European Commission 2006, 2007). Analytical methods for rapid, sensitive, and accurate determination of these mycotoxins in unprocessed cereals and cereal-based products are highly needed in order to properly assess both the relevant risk of exposure and the relevant toxicological risk for humans and animals, as well as to ensure that regulatory levels fixed by the EU or other international organisations are met. Analytical methods for mycotoxins in cereals and cereal-based products generally require toxin extraction from the matrix with an adequate extraction solvent, a clean-up step intended to eliminate interference from the extract and, finally, detection/determination of the toxin by suitable analytical instruments/ technologies. Chromatographic methods commonly used for quantitative determination of mycotoxins in cereals include high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV), diode array (DAD), fluorescence (FD) or mass spectrometry (MS) detectors, and gas-chromatography (GC) coupled with electron capture (ECD), flame ionization (FID) or MS detectors. In addition, commercial immunometric assays, such as enzyme-linked immunosorbent assays (ELISA) or membrane-based immunoassays, are frequently used for screening purposes. Recently, a variety of rapid methods that are emerging have been proposed for mycotoxin analyses. They are based on novel technologies, including immunochromatography, fluorescence polarization, infrared spectroscopy, molecularly imprinted polymers, and biosensors (S h e p h a r d, 2008). This review deals with the current analytical methodologies, traditional and emerging, available for the detection of main mycotoxins occurring in cereals and cereal-based products. Sample preparation (extraction and clean-up) Mycotoxins are commonly extracted from ground cereals by shaking or blending with mixtures of water or other polar solvents, such as methanol or acetonitrile. Purification of the extract is an essential step in the analysis of mycotoxins, especially when chromatographic techniques are used for their determination at trace levels. Solid phase extraction (SPE), multifunctional clean-up columns, e.g. MycoSep, and immunoaffinity columns (IACs) are frequently used to clean-up the extracts of raw cereals, as well as cereal-processed products. MycoSep columns are one of the most commonly used and commercially available columns for removing analytical interferences from raw extracts in one quick step (1030 sec). The MycoSep column contain16

ing various adsorbents, such as charcoal, Celite and ion-exchange resins, is pushed into a test tube (containing the extract) forcing the extract to filter upwards through the packing adsorbent material. The interferences adhere to the adsorbents in the column and the purified extract passes through a frit to the surface of the column. These columns are often used for the simultaneous and rapid clean-up of type A- and type B-trichothecenes, as well as AFs, OTA, ZEA and FBs. Immunoaffinity columns are based on monoclonal or polyclonal antibodies, and are commonly used for mycotoxin analyses. The specificity of antibodies provides cleaner extracts, with respect to other methods of purification, and good precision, accuracy and sensitivity of analytical methods that use this clean-up procedure. IACs are commercially available for AFs, OTA, FBs, ZEA, DON, T-2 and HT-2 toxins, and have been used to simultaneously detect the presence of these toxins by HPLC with good accuracy and precision (P a s c a l e and V i s c o n t i, 2008). Recently, in the area of mycotoxin analysis, there has been an increasing interest in the potential use of molecularly imprinted polymers (MIPs) as adsorbents for SPE due to their low costs, easy preparation, high chemical stability and long shelf-life. MIPs are cross-linked polymers that are thermally, photochemically or electrochemically synthesized by the reaction of a monomer and a cross-linker in the presence of an analyte, e.g. mycotoxin, used as a template. After polymerization, the analyte is removed leaving specific recognition sites inside the polymer. MIPs provide biomimetic recognition elements capable of selective binding/rebinding to the analyte with efficiencies comparable to those of antibody-antigen interactions. The synthesis of MIPs with high affinity for DON, ZEA and OTA was already reported (P a s c a l e et al., 2008a). These polymers have been used as a stationary phase in chromatographic applications, or for the preparation of SPE columns that are to be used in sample clean-up, although, in a few cases, non-imprinted polymers, i.e. polymers synthesized without a mycotoxin template, performed similarly to molecularly imprinted polymers. Recently, itaconic acid has been identified by molecular modeling and computational design as a functional monomer with high affinity towards DON. Itaconic acid polymers, synthesized without the template (i.e. DON), were successfully used as adsorbents for SPE clean-up and pre-concentration of DON from pasta extracts prior to the HPLC analysis (P a s c a l e et al., 2008a). Traditional technologies for detecting/quantifying mycotoxins Gas chromatographic methods based on FID, ECD and MS detection are the most widely used methods for quantitative simultaneous determination of trichothecenes (mainly type A) in cereals and cereal-based products (K r s k a et al., 2001). These methods require a preliminary clean-up of extracts, generally by MycoSep columns, and pre-column derivatization of the purified extract with specific reagents. Mass spectrometry (MS), or tandem mass spectrometry (MS/MS), offers an advantage in confirming the identity of chromatographic peak. The main problems associated to GC analysis include increa17

sed trichothecene responses (up to 120%), non-linearity of calibration curves, drifting responses, carry-over or memory effects from previous samples, and high variation in terms of reproducibility and repeatability (P e t t e r s o n and L a n g s e t h, 2002). HPLC coupled with UV, diode array (DAD) or fluorescence detector (FD) is currently the most widely used technique for the analysis of major mycotoxins occurring in cereals. AFs, OTA, FBs and ZEA are routinely analyzed by HPLC-FD, and DON by HPLC-UV (DAD) with good accuracy and precision. HPLC-FD is highly sensitive, selective and repeatable, so specific labeling reagents have been developed for the derivatization of non-fluorescent mycotoxins to form fluorescent derivatives. Either pre-column derivatization with trifluoroacetic acid (TFA), or post-column derivatization with Kobra Cell (i.e. electrochemical bromination cell) or photochemical reactor (i.e. UV irradiation), can be used to enhance fluorescence of aflatoxins B1 and G1 (P a p a d o p o u l o u - B o u r a o u i et al., 2002), whereas pre-column derivatization with OPA reagent is required for the detection of fumonisins B1, B2 and B3, after purification of the extracts with immunoaffinity columns, solid phase extraction or MycoSep columns. Recently, 1-anthroylnitrile (1-AN), 2-naphthoyl chloride (2-NC), and pyrene-1-carbonyl cyanide (PCC) have been used as fluorescent labeling reagents for T-2 and HT-2 detection by HPLC-FD (V i s c o n t i et al., 2005; L i p p o l i s et al., 2008). The derivatization reaction was used to develop a sensitive, reproducible and accurate method for the simultaneous determination of T-2 and HT-2 after IAC clean-up in naturally contaminated cereal grains (wheat, maize, oats and barley). Several HPLC methods for identifying various mycotoxins in a number of cereals and cereal-based products have been validated by collaborative studies, and their performance characteristics, such as accuracy, repeatability, reproducibility, detection and quantification limits were established. These methods have been adopted as official or standard methods by the AOAC International or the European Standardization Committee (CEN). In particular, methods for measuring aflatoxins in maize (AOAC Official Method 991.31 and 2005.08), ochratoxin A in barley (2000.03), aflatoxin B1 in baby food (2000.16), fumonisins B1 and B2 in maize flour and cornflakes (2001.04) that use IACs clean-up and HPLC-FD are approved as official methods by AOAC International (http://www.aoac.org). In addition, HPLC/IAC methods have been validated for the measurement of DON in cereals and cereal products, and ZEA in barley, maize, wheat flour, polenta and maize-based baby food (M a c D o n a l d et al., 2005a; M a c D o n a l d et al., 2005b). Liquid chromatography coupled with mass spectrometry (LC-MS) has been used for many years, mainly as a technique for mycotoxin confirmation. At the present time, LC-MS and LC-MS/MS are the most promising techniques for the simultaneous screening, identifying and measuring of a large number of mycotoxins. Advances and recent trends in mycotoxin detection by LC-MS have been recently reviewed (S f o r z a et al., 2006; S o n g s e r m s a k u l and R a z z a z i - F a z e l i, 2008). The following mycotoxins were examined: patulin, aflatoxins, ochratoxin A, zearalenone and its metabolites, trichothecenes and fumonisins. LC-MS/MS and Atmospheric Pressure Chemi18

cal Ionization (APCI) or Electro-Spray Ionization (ESI) interface was used for the simultaneous determination of the major type A- and type B-trichothecenes and ZEA in cereals and cereal-based products at trace levels (B e r t h i l l e r et al., 2005). LC-APCI-MS/MS method using reversed phase SPE Oasis HLB columns for extract clean-up has been recently developed for the simultaneous determination of NIV, DON, T-2 and HT-2 in cereals and cereal-based products (L a t t a n z i o et al., 2008). The method was applied to a large number of cereals (wheat, barley and maize) and cereal-based foods (infant semolina, infant biscuits, bacon biscuits, cocoa wafers and coconut snacks) with detection limits in the g/kg range. LC-ESI-MS/MS method has been recently developed for the simultaneous determination of major Fusarium toxins (DON, T-2, HT-2, FB1, FB2, ZEA) together with aflatoxins (AFB1, AFB2, AFG1, AFG2) and OTA in maize, based on the use of new multi-toxin immunoaffinity columns, i.e. Mycodin1 (L a t t a n z i o et al., 2007). HPLC-MS/MS is also proved to be a powerful technique for the determination of masked mycotoxins, for example deoxynivalenol-glucosides, in wheat (B e r t h i l l e r et al., 2007). Masked mycotoxins are mycotoxins conjugated to more polar substances, e.g. glucose, not detected by routine analytical methods, even though they can release their toxic precursors after hydrolysis. Accuracy, precision, and sensitivity of LC-MS methods may vary depending on the mycotoxin, matrix and instrument with the sensitivity of the method depending on the ionization technique used. Quantitative measurement of mycotoxins by LC-MS is often unsatisfactory due to matrix effects and ion suppression. Purification of extracts by MycoSep or IACs is generally needed prior to MS detection (L a t t a n z i o et al., 2009). Immunological assays, such us ELISA, have become very popular in mycotoxin screening since the late 1970s (P e s t k a et al., 1995). In general, ELISA does not require clean-up procedures, and the extract containing the mycotoxin is analyzed directly. Even though they often lack accuracy at very low concentrations and are limited in the range of matrices examined, immunoassays provide fast, inexpensive screening assays. However, matrix interference or the presence of structurally related mycotoxins can interfere with the binding of conjugate and antibody, leading to mistakes in quantitative measurements of mycotoxins. ELISA kits should be used routinely only for the analysis of matrices that are extensively tested. Confirmatory analyses by more robust methods, e.g. HPLC with IAC clean-up or LC-MS, are required for the contamination levels that approach the legal limit. Several ELISA kits that use monoclonal or polyclonal antibodies against mycotoxins have been commercially developed for qualitative, semi-quantitative or quantitative analyses of the main known mycotoxins in cereal-based matrices. Some ELISAs have been validated by collaborative studies and adopted by the AOAC International as official methods for determination of aflatoxin B1/total aflatoxins and ZEA in food and feed matrices (AOAC Official Methods No. 989.06, No. 990.32, No. 990.34, No. 991.45, No. 993.16 and No. 994.01). Nevertheless, the use of ELISAs to detect mycotoxins at contamination levels approaching the legal limits is inappropriate since these assays were validated at levels much higher than the legal limits. Direct ELISA has been validated for measu19

ring total fumonisins (i.e. sum of FB1, FB2 and FB3) in maize at levels > 1.0 mg/kg (AOAC Official Methods No. 2001.06) with good precision. Emerging technologies for mycotoxin analysis Over the past years, rapid immunoassay-based tests have increasingly been used for the analyses of mycotoxins in cereals and cereal-based foods (Z h e n g et al., 2006; G o r y a c h e v a et al., 2007). Lateral flow devices (LFDs), also called immunochromatographic strip tests, are rapid immunoassays based on the interaction between specific antibodies, immobilized on a membrane strip, and antibody-coated dyed receptors, e.g. latex or colloidal gold, that react with analyte to form an analyte-receptor complex. LFDs have been developed for the most prevalent mycotoxins in cereals and are commercially available for the determination of AFs and FBs in maize, DON in wheat, and OTA, ZEA, T-2 and HT-2 in cereal grains. Portable photometric strip readers allow quantitative or semi-quantitative analysis (K r s k a and M o l i n e l l i, 2009). Fluorescence polarization immunoassay (FPIA) is a simple technique that measures interactions between a fluorescently labeled antigen and a specific antibody in solution. FPIAs are developed for rapid determination of AFs, ZEA, FBs and DON, although low accuracy and sensitivity were observed when these assays were used with cereal samples (N a s i r and J o l l e y, 2003; M a r a g o s and P l a t t n e r, 2002; C h u n et al., 2009). Recently, FPIA has been optimized for rapid determination of DON in durum and common wheat, semolina and pasta (L i p p o l i s et al., 2006; P a s c a l e et al., 2008b). The assay showed better accuracy and precision, with respect to a widely used HPLC-IAC method (M a c D o n a l d et al., 2005a), in the range of 1002.000 mg/kg. Fourier transform near-infrared (FT-NIR) spectroscopy and principal component analysis (PCA) have been recently used for the determination of DON content in durum and common wheat. A semi-quantitative model was developed to discriminate between blank and naturally contaminated wheat samples at 300 mg/kg (De G i r o l a m o et al., 2009). A modified approach to evaluate DON content in scab-damaged wheat by using diffuse reflectance UV-vis spectroscopy was reported (S i u d a et al., 2008). The applicability of NIR reflectance spectroscopy, combined with multivariate statistical methods, was evaluated for its ability to predict the incidence of fungal infection in maize and fumonisin B1 content by analyzing 280 naturally and artificially contaminated maize samples, although the limit of detection of the method was not reported (B e r a r d o et al., 2005). The advantages of these methods compared to other methods are the ease of operations, rapidity of analysis and non-destruction of samples. Immunochemical biosensors that use surface plasmon resonance (SPR) or screen-printed carbon electrodes have been described for the detection of mycotoxins in cereals. Competitive SPR-based immunoassays have been recently described for the determination of DON in wheat, with or without 20

extract clean-up, showing a good correlation between DON concentration measured with biosensor and GC-MS, or HPLC-UV and HPLC-MS/MS as reference methods (P r i e t o - S i m o n et al., 2007). Competitive electrochemical ELISAs based on disposable screen-printed carbon electrodes have been developed for quantitative determination of ochratoxin A in wheat. The results from screen-printed carbon electrodes and HPLC/IAC clean-up methods for naturally contaminated wheat samples showed good correlation (A l a r c n et al., 2006). CONCLUSION Several analytical methods for the determination of major mycotoxins occurring in cereals and cereal-based products have been developed and continuously improved. Advantages and disadvantages of traditional and emerging methods are reported in Table 1. Among the traditional methods, immunoaffinity/MycoSep column clean-up coupled with HPLC is the most frequently used technique for the measurement of main mycotoxins occurring in cereals and cereal-based products, although LC-MS/MS seems to be the most promising technique to be used in the future for multi-mycotoxins analysis. Various immunological methods, ELISA and other rapid antibody-based tests, are generally used for screening purposes, although these methods often require confirmatory analyses with more robust methods. Finally, few emerging technologies, which are often combined with immunochemical assays, have been proposed for rapid analysis of mycotoxins in cereals. Among these, FP immunoassay has the potential for reliable high-throughput analysis of DON in wheat and wheat-based products.
Tab. 1 Advantages and disadvantages of traditional and emerging methods for mycotoxin analysis Method GC Advantages Simultaneous analysis of mycotoxins, good sensitivity, may be automated (autosampler), provides confirmation (MS detector). Disadvantages Expensive equipment, specialist expertise required, derivatization required, matrix interference problems, non-linear calibration curve, drifting response, carry-over effects from previous sample, variation in reproducibility and repeatability. Expensive equipment, specialist expertise required, may require derivatization.

HPLC

Good sensitivity, good selectivity, good repeatability, may be automated (autosampler), short analysis times, official methods available. Simultaneous analysis of mycotoxins, good sensitivity (LC/MS/MS), provides confirmation, no derivatization required.

LC/MS

Very expensive, specialist expertise required, sensitivity relies on ionization technique, matrix assisted calibration curve (for quantitative analysis), lack of internal standards.

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ELISA

Simple sample preparation, inexpensive equipment, high sensitivity, simultaneous analysis of multiple samples, suitable for screening, limited use of organic solvents. Rapid, no clean-up, no expensive equipment, easy to use, no specific training required. Rapid, no clean-up required, validated for DON in wheat.

Cross-reactivity with related mycotoxins, matrix interference problems, possible false positive/negative results, confirmatory LC analysis required. Semi-quantitative (visual assessment), cross-reactivity with related mycotoxins, validation required for additional matrices. Inconsistent with ELISA or HPLC analyses (except for DON), poor sensitivity in some cases, cross-reactivity with related mycotoxins, matrix interference problems. Expensive equipment, calibration model must be validated, knowledge of statistical methods, poor sensitivity. Cross-reactivity with related mycotoxins, extract clean-up needed to improve sensitivity, variation in reproducibility and repeatability. Poor selectivity.

LFD

FPIA

IR spectroscopy

Rapid, non-destructive measurement, no extraction or clean-up, easy operation. Rapid, no clean-up procedure.

Biosensors

MIP

Low cost, stable, reusable.

GC = Gas Chromatography; HPLC = High Performance Liquid Chromatography; LC/MS = Liquid Chromatography/Mass Spectrometry; ELISA = Enzyme-Linked Immunosorbent Assay; LFD = Lateral Flow Device; FPIA = Fluorescence Polarization Immunoassay; IR = Infrared; MIP = Molecularly Imprinted Polymer.

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METODE DETEKCIJE MIKOTOKSINA U ITARICAMA I PROIZVODIMA OD ITA Mikelanelo Paskale Institut za prehrambenu proizvodwu, Nacionalni istraivaki savet G. Amendola 122/0, 70126 Bari, Italija Rezime Metode analize mikotoksina u itaricama i proizvodima od ita se sastoje iz tri postupka: ekstrakcije, preiavawa (kako bi se uklonile interferencije ekstrakta i koncentrisao analit) i detekcije/odreivawa toksina (koriewem odgovarajuih analitikih instrumenata/tehnologije za analizu). Preiavawe je kquni postupak u analizi mikotoksina pri wegovim koncentracijama u tragovima koji obuhvata primenu ekstrakcije vrstom fazom i multifunkcionalne (npr. MycoSep) ili imunoafinitetne kolone. Razliite hromatografske metode se koriste za kvantitativno odreivawe mikotoksina, ukquujui gasnu hromatografiju (GC) uz koriewe detektora sa zahvatom elektrona, plameno jonizacione detektore ili masenu spektrometriju (uglavnom za trihotecene tipa A) kao i tenu hromatografiju visokog uinka (HPLC) uz koriewe UV, DAJD, fluorescentnih i MS detektora. Izbor metode zavisi od matriksa i mikotoksina koji se analizira. Tena hromatografija u paru sa masenom spektrometrijom (LC-MS/MS) je tehnika koja najvie obeava u sluaju simultanog skenirawa, identifikacije i kvantitativnog odreivawa velikog broja mikotoksina. Osim toga, komercijalna imunometrijska ispitivawa, kao to je enzimski imunosorpcioni test (ELISA), takoe se esto koriste u svrhe skenirawa. U posledwe vreme velik broj metoda koje se baziraju na novim tehnologijama mogu se koristiti za analizu mikotoksina, a meu wima su imunohromatografija (npr. lateralni protoni ureaji), imuno testovi sa polarizacijom fluorescentne emisije (FPIA), infracrvena spektroskopija (FT-NIR), polimeri sa molekularnim otiskom (MIPs) i optiki biosenzori.

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