Thin Layer Chromatography Molicular Imprinted Printed Testing Rapid

Reusable

Poor selectivity

LFD = Lateral Flow Device; FPIA = Fluorescence Polarization Immunoassay; IR = Infrared Spectroscopy (NIR, near-infrared; MIR, mid-infrared); Immunosensors/Biosensors = Surface Plasmon Resonance (SPR); Fiber Optic Immunosensors (FOI); Quartz Crystal Microbalance (QCM); Screen-Printed Carbon Electrodes (SPCE); MIP = Molecularly Imprinted Polymer.

LFD = Lateral Flow Device; FPIA = Fluorescence Polarization Immunoassay; IR = Infrared Spectroscopy (NIR, near-infrared; MIR, mid-infrared); Immunosensors/Biosensors = Surface Plasmon Resonance (SPR); Fiber Optic Immunosensors (FOI); Quartz Crystal Microbalance (QCM); Screen-Printed Carbon Electrodes (SPCE); MIP = Molecularly Imprinted Polymer.

Classical technologies for detecting/quantifying mycotoxins Thin-layer chromatography (TLC)

TLC is a simple, cost-effective technique often used as a mycotoxin screening assay when low detection limits are not required. A paper highlighting the status of TLC methods for my-cotoxins in various sample matrices was published in a special issue of the Journal of Chro-matographyA (Lin etal., 1998). Although TLC is a powerful tool for the simultaneous analysis of multiple samples for multiple mycotoxins, it cannot be used for sensitive or precise measurements unless densitometric analyses are performed. Highly reproducible, reliable re sults can be obtained if an autospotter is used to apply the samples. TLC can be used without cleaning up the extract, but extract purification prior to spotting increases sensitivity (Stroka et al., 2000). A reversed-phase TLC method has been validated for the measurement of fumo-nisin Bi in maize at ^g/g levels (Shephard and Sewram, 2004), and a two-dimensional high performance thin layer chromatography (HPTLC) method has been developed for the determination of ochratoxin A at 5 ng/g in green coffee beans (Ventura et al., 2005).

Gas chromatography (GC)

Gas chromatographic methods based on flame ionization detection (FID), electron-capture detection (ECD) and MS detection are the most widely used methods for quantitative determination of trichothecenes (mainly type A) in foods and feedstuffs (Krska et al., 2001). These methods require preliminary clean-up of extracts by charcoal-alumina, Florisil®, silica gel or MycoSep® columns and pre-column derivatization of the purified extract with specific reagents to increase the volatility and sensitivity of the toxins. A comparative inter-laboratory study, funded by the European Union, evaluated the performance of methods for analyzing trichothecenes with GC and found that improvements in methods are needed in toxin recovery and in the accuracy and precision of the measurements. The main problems came from compounds in the matrix that increased the trichothecene response (up to 120%) and resulted in non-linear calibration curves, drifting responses, carry-over or memory effects from previous samples, and high variation in terms of reproducibility and repeatability (Petterson and Langseth, 2002).

High performance liquid chromatography (HPLC)

HPLC coupled with UV, a diode array detector (DAD) or a fluorescence detector (FD) currently is the most widely used technique for the identification of the major mycotoxins in food commodities. Aflatoxin M1, ochratoxin A, zearalenone, patulin and deoxynivalenol are routinely analyzed by HPLC/FD or HPLC/UV(DAD) with good accuracy and precision. HPLC/FD is highly sensitive, selective and repeatable, so specific labeling reagents have been developed, and are commercially available, for the derivatization of non-fluorescent mycotoxins to form fluorescent derivatives. Either pre-column derivatization, with trifluoroacetic acid (TFA), or post-column derivatization, with Br or I, can be used to identify aflatoxins B1, B2, G1 and G2, whereas pre-column derivatization with OPA reagent is required for fumonisins B1, B2 and B3, and T-2 and HT-2 toxins require pre-column derivatization with 1-anthroylnitrile after purification of the extracts with immunoaffinity columns, solid phase extraction or MycoSep® columns.

Several HPLC methods for identifying various mycotoxins in a number of foods have been validated by collaborative studies, for which performance characteristics such us 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. In particular, methods for measuring aflatoxins in maize, raw peanuts and peanut butter (AOAC Official Method 991.31), aflatoxin B1 and total aflatoxins in peanut butter, pistachios, figs and paprika (999.07), ochratoxin A in barley (2000.03), aflatoxin M1 in milk (2000.08), aflatoxin B1 in baby food (2000.16), ochratoxin A in roasted coffee (2000.09), ochratoxin A in wine and beer

(2001.01), fumonisins Bi and B2 in maize flour and maize flakes (2001.04), aflatoxins in animal feed (2003.02) and ochratoxin A in green coffee (2004.10) that use immunoaffinity column clean-up and HPLC/FD have been approved as official methods by AOAC International (http://www.aoac.org). In addition, HPLC/immunoaffinity column methods have been validated for the measurement of deoxynivalenol in cereals and cereal products, zearale-none in barley, maize, wheat flour, polenta and maize-based baby food, aflatoxins in hazelnut paste and ochratoxin A in cocoa powder (Brera et al., 2005; MacDonald et al., 2005a,b; Senyuva and Gilbert, 2005).

Liquid chromatography/mass spectrometry (LC/MS)

Liquid chromatography coupled with mass spectrometry has been used for many years mainly as technique for mycotoxin confirmation. At the present time, LC/MS is the most promising technique for simultaneously screening, identifying and measuring a large number of mycotoxins.

Advances in mycotoxin detection by hyphenated chromatographic techniques/mass spectrometry have been reviewed recently (Sforza et al., 2006). The following mycotoxins were examined: patulin, aflatoxins, ochratoxin A, zearalenone and its metabolites, tii-chothecenes and fumonisins. HPLC with tandem mass spectrometry (HPLC-MS/MS) and an Atmospheric Pressure Chemical Ionization or Electro-Spray Ionization interface was used for the simultaneous determination of the major type A- and type B-trichothecenes and zearalenone in cereals and cereal-based products at trace levels (Berthiller et al., 2005a). HPLC-MS/MS also has been used for the simultaneous identification of nine mycotoxins -aflatoxins B1, B2, G1, G2 and M1, ochratoxin A, mycophenolic acid, penicillic acid and roquefortine C - in cheese, for the identification of 18 mycotoxins and metabolites - ochratoxin A, zearalenone, a-zearalenol, p-zearalenol, a-zearalanol, p-zearalanol, fumonisins B1 and B2, T-2 toxin, HT-2 toxin, T-2 triol, diacetoxyscirpenol, 15-monoacetoxyscirpenol, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, deepoxy-deoxynivalenol and aflatoxin M1 - in milk, and for the determination of zearalenone and its metabolites in various biological matrices (Kleinova et al., 2002; Kokkonen et al., 2005; Sorensen and Elbaek, 2005). HPLC-MS/MS also is a powerful technique for the determination of masked deoxynivalenol, i.e., deoxynivalenol-glucosides, in wheat. 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 (Berthiller et al., 2005b).

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 often is unsatisfactory due to matrix effects and ion suppression. Purification of extracts by MycoSep® or immunoaffinity columns generally is needed prior to MS detection.

Enzyme-linked immunosorbent assays (ELISAs)

Immunological assays have been used to successfully detect mycotoxins since the late 1970s (Pestka et al., 1995). Several microtiter plate- or membrane-based ELISAs that use monoclonal or polyclonal antibodies against mycotoxins currently are available commercially for qualitative, semi-quantitative and quantitative analysis of the major known myco-

toxins from a number of food matrices. In general, ELISAs do not require clean-up procedures and the extract containing the mycotoxin is analyzed directly. Even though they often lack accuracy at very low concentrations (competitive assays) 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 binding of the conjugate and the antibody leading to mistakes in quantitative measurements of mycotoxins. ELISA kits should be used routinely only for the analysis of matrices that have been extensively tested. Confirmatory analyses by more robust methods, e.g., HPLC with an immunoaffinity column clean up or LC-MS, are required for contamination levels that approach the legal limit.

Some immunoassays have been validated by collaborative studies and adopted by the AOAC International as official methods for determination of aflatoxin Bi/total aflatoxins and zearalenone 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. In addition, ELISAs have less precision than either TLC or HPLC methods. A direct ELISA has been validated for measuring total fumonisins, i.e., fumonisins B1 + B2 + B3, in maize at levels > 1.0 |ig/g (AOAC Official Methods No. 2001.06) with good precision.

Flow-through enzyme immunoassays for field use have been developed for the rapid detection of aflatoxin B1, aflatoxin M1, fumonisins, ochratoxin A, zearalenone and T-2 toxin. A quick qualitative (visual) discrimination between positive and negative samples can be made at levels close to the regulatory limits. Rapid, flow-through tests have been validated for several matrices and provide results consistent with those obtained by HPLC. This assay was both accurate and reliable giving no false compliant and only a few false non-compliant results (Papens et al., 2004).

The U.S. Department of Agriculture - Grain Inspection, Packers and Stockyards Administration (USDA-GIPSA) has implemented a program (GIPSA Directive 9181.2) to verify the performance of antibody-based rapid kits for the quantification of mycotoxins in grain and grain products (http://archive.gipsa.usda.gov/reference-library/directives/9181-2.pdf). A number of kits for aflatoxins, deoxynivalenol and fumonisins have been verified and selected (http://archive.gipsa.usda.gov/tech-servsup/metheqp/testkits.pdf).

Emerging technologies for mycotoxin analyses Lateral flow devices (LFDs)

A lateral-flow device, also called an immunochromatographic test, is a rapid immunoassay 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 the analyte to form an analyte-receptor complex. Competitive LFDs rely on the competition of the analyte, e.g., a mycotoxin, in solution for the binding sites of the labeled receptor. The test line contains an analyte-conjugate attached to the membrane that binds unbound receptor to form a colored analyte-conjugate receptor complex. The control line includes a specific antibody attached to the membrane that binds with the labeled receptor. Upon binding, the control line changes to a colored signal. If the signal in the test line is missing or weakly visible, the test indicates that the analyte is present in a sufficient amount (positive test). If the test line signal is clearly visible, the test indicates that the analyte is not present in the extract (negative test). The benefits of LFDs include user-friendly format, rapid response and price. These features make strip tests ideal for applications such as "on-site" detection of environmental and agricultural analytes.

A one-step LFD has been developed in a competitive immunoassay format, for the determination of ochratoxin A in fungal cultures. The use of an immunoaffinity column cleanup of grain extracts allowed LFDs to be used to determine the presence of ochratoxin A at low levels (Danks et al., 2003).

Lateral flow immunochromatographic assays are commercially available for the determination of aflatoxin Bi in maize and peanuts (Rosa®aflatoxin - Charm, USA; AgraStrip - Romer Labs, USA; Reveal®aflatoxin - Neogen, USA), aflatoxin Mi in milk (Ro-sa®aflatoxin Mi - Charm, USA) and DON in wheat (RIDA®Quick DON - Biopharm, Germany; Reveal®DON - Neogen, USA). The Rosa®aflatoxin test was the first quantitative lateral flow test developed that was approved by USDA-GIPSA for determination of anatoxins in maize samples at levels > i.0 ng/g.

Fluorescence polarization immunoassay (FPIA)

FPIA is a simple technique that measures interactions between a fluorescently labeled antigen and a specific antibody. The technology, first developed in the 1970s, has long been used in human and veterinary diagnostics. Fluorescence polarization is based on differences in the rate of rotation between a free fluorescently labeled antigen and the same antigen bound to a specific antibody. Adaptation of these assays to mycotoxin analyses is still in progress. FPIAs have been used for the rapid determination of aflatoxins, zearalenone, fu-monisins and deoxynivalenol in solution, although low accuracy and sensitivity were problems when these assays were used with cereal samples (Maragos and Kim, 2004). Recently, an optimized FPIA has been developed for rapid screening of deoxynivalenol in wheat and derivative products (pasta and semolina). In a comparison with a widely used HPLC/immunoaffinity method, FPIA was a rapid, inexpensive alternative to the more robust chromatographic methods for the determination of deoxynivalenol in wheat and derivative products (Lippolis et al., 2006).

Infrared spectroscopy

The use of near-infrared (NIR) transmittance spectroscopy and mid-infrared (MIR) spectroscopy with attenuated total reflection for the rapid determination of deoxynivalenol in kernels of wheat and maize has been evaluated by observing changes in protein, lipid and carbohydrate contents. Principal component analyses, and cluster analyses or partial least square regression models were used to identify samples contaminated with > 310 ng/g deoxynivalenol by MIR and to predict deoxynivalenol content at levels close to the legal limit in the European Union, i.e., 1,750 ng/g, by NIR. The applicability of NIR reflectance spectroscopy, combined with multivariate statistical methods, was recently 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. The NIR methodology was used to monitor mold contamination in post-harvest maize and to distinguish lots contaminated with fumonisin Bi from those that were not contaminated, although the limit of detection of the method was not reported (Berardo et al, 2005).

Capillary electrophoresis

Capillary electrophoresis is an analytical technique that allows good separation of mycotox-ins from potential interfering species present in the extract on the basis of electrical charge. Capillary electrophoresis methods have been developed for various mycotoxins, including aflatoxins, citrinin, deoxynivalenol, fumonisins, moniliformin, ochratoxins, penicillic acid, roridin A, sterigmatocystin, and zearalenone by using UV/visible detection. Recently, fluorescence-based capillary electrophoresis methods have been developed for several myco-toxins allowing their detection at levels commonly found in naturally contaminated food samples. Capillary zone electrophoresis with laser-induced fluorescence was used to measure ochratoxin A in roasted coffee, maize and sorghum after tandem clean-up (silica and immunoaffinity columns) of extracts for the analysis of aflatoxin Bi in maize, and for the measurement of fumonisin Bi in maize after immunoaffinity column clean-up and derivati-zation with fluorescein isothiocyanate (Corneli and Maragos, 1998). A capillary zone elec-trophoresis-diode array detection method was developed for the measurement of moniliformin in maize. Capillary zone electrophoresis methods are comparable in sensitivity, precision and accuracy to HPLC methods (Maragos, 2004). The use of less expensive capillaries, the absence of organic solvents during the detection step, and shorter analysis times, all make capillary-zone electrophoresis methods viable alternatives to those requiring HPLC.

Fiber-optic immunosensors

Evanescent wave-based fiber-optic immunosensors have been developed for the detection of fumonisin B1 and aflatoxin B1 in maize. A competitive format was used to measure fumonisin B1 in solution by immobilizing fumonisin monoclonal antibodies on an optical fiber and measuring the competition between fumonisin B1 and a fumonisin B1 fluorescently labeled probe for binding to the fiber. A detection limit of 3.2 ^g/g was observed. The use of an immunoaffinity column for clean-up of the extracts increased the sensitivity of the method (detection limit 0.4 ^g/g). A non-competitive assay was used for aflatoxin B1 that takes advantage of the native fluorescence of this mycotoxin. The sensor could detect 2 ng/ml of aflatoxin Bi in phosphate buffered saline solution. Problems due to refractive index-related effects were observed in the presence of organic solvents, which reduced the specificity of the assay (Maragos and Thompson, 1999).

Biosensors

A biosensor is an analytical device that incorporates a specific biological element, e.g., an antibody, that creates a recognition event and a physical element that transduces the recognition event into an acoustic, electrical or optical signal. Immunochemical biosensors that use surface plasmon resonance, quartz crystal microbalance and screen-printed carbon electrodes have been described for the detection of mycotoxins.

Competitive surface plasmon resonance-based immunoassays have been used for rapid screening of aflatoxin B1, zearalenone, ochratoxin A, fumonisin B1 and deoxynivalenol in naturally contaminated matrices (Daly et al. 2000; Dunne et al, 2005; Tudos et al, 2003; van der Gaag et al., 2003). Surface plasmon resonance biosensors measure refractive index changes that occur on a metal film and provide a signal that is positively correlated with the mass density changes on the metal surface. Mycotoxins were chemically modified and then immobilized on the surface of the sensor chip. A fixed concentration of specific antibody is mixed with sample extract containing the mycotoxin to be detected and the mixture is passed over the sensor surface where non-conjugated antibodies are bound to the mycotox-in on the surface. Regeneration of the surface of the sensor chip allows reuse of the same chip up to 100 times. All toxins were detected at very low levels with good accuracy and precision. The use of a surface plasmon resonance equipment with four flow cells enables the detection of four mycotoxins in a single measurement (van der Gaag et al, 2003).

A quartz crystal microbalance biosensor has been developed for the rapid measurement of ochratoxin A in liquid matrices, e.g., water or juices, at concentrations ranging from 4 to 50 ng/ml without a clean-up step (Visconti and de Girolamo, 2005).

Competitive electrochemical ELISAs based on disposable screen-printed carbon electrodes have been developed for quantitative determination of ochratoxin A in wheat and in wine. Detection limits were 0.4 ng/g and 0.9 ng/ml, respectively. Results from screen-printed carbon electrodes and HPLC/immunoaffinity column clean-up methods for naturally contaminated samples were correlated (Alarcon et al., 2006).

All of these biosensors have the potential to be very convenient in terms of time of analysis, but may require coupling with a clean-up technique for optimal effectiveness.

Molecularly imprinted polymers

Molecularly imprinted polymers are cross-linked polymers that are thermally, photochemi-cally or electrochemically synthesized by the reaction of a monomer, e.g., methacrylic acid, 4-vinyl pyridine or pyrrole, and a cross-linker, e.g., ethylene glycol dimethacrylate, divinyl benzene, in the presence of an analyte, e.g., a mycotoxin, or mimic compounds, i.e., a "dummy", used as a template. After polymerization, the analyte is removed leaving specific recognition sites inside the polymer. Molecularly imprinted polymers provide biomimetic recognition elements capable of selective binding/rebinding to the analyte with efficiencies comparable to those of antibody-antigen interactions. The development of molecularly imprinted polymers for mycotoxins is very attractive due to their low costs, easy preparation, high chemical stability and long shelf-life. The synthesis of molecularly imprinted polymers with high affinity for deoxynivalenol, zearalenone and ochratoxin A has been reported (Weiss et al., 2003; Zhou et al., 2004; Visconti and de Girolamo, 2005; Yu et al., 2005). These polymers have been used as the stationary phase in chromatographic applications or for the preparation of solid phase extraction columns for use 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, a molecularly imprinted polypyrrole film was synthesized on the sensor of a miniaturized surface plasmon resonance device for detection of ochratoxin A in wheat (Yu and Lai, 2005).

Conclusions

Several analytical methods for the measurement of mycotoxins occurring in foods, feeds and beverages have been developed and continuously improved in order to satisfy performance criteria (accuracy and precision) and to reliably reach the low detection limits needed for risk assessment studies. Currently, immunoaffinity column clean-up coupled with HPLC is the most powerful technique for the measurement of the major known mycotoxins occurring in agricultural and food commodities. HPLC/immunoaffinity column methods have good sensitivity and comply with the standards established by international organizations, e.g., AOAC International and the European Standardization Committee. A number of official/validated methods based on HPLC/FD or UV and immunoaffinity column clean-up are currently available for the determination of the major mycotoxins in several food matrices that fulfill European Union and other international regulations. Various immunological assays, both ELISAs and other rapid antibody-based tests, also are available for measuring mycotoxins in different foods and generally used for screening purposes. These methods often require confirmatory analyses by HPLC/immunoaffinity columns or LC-MS. Methods for the simultaneous detection of multiple mycotoxins are highly desirable for screening purposes. HPLC coupled with mass spectrometry detection seems to be the most promising technique for the simultaneous determination and identification of a large number of mycotoxins. Finally, several novel technologies, which often are combined with im-munochemical assays, have been proposed for the rapid analysis of mycotoxins in foods and beverages, but further investigations are required to validate them and to determine their applicability to real samples, especially at levels close to legal limits.

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