WO2000042421A1

WO2000042421A1 - Method for screening for type b trichothecene mycotoxins

Info

Publication number: WO2000042421A1
Application number: PCT/US2000/001049
Authority: WO
Inventors: Michael S. Freund; Chen-Chan Hsueh; Yi Liu
Original assignee: Competitive Technologies, Inc.
Priority date: 1999-01-15
Filing date: 2000-01-15
Publication date: 2000-07-20

Abstract

This invention relates to a method for screening a sample for type B trichothecene mycotoxins and, more particularly, to electrochemical detection of type B tricothecene mycotoxins contamination from the natural growth of fungi such as in agricultural products as well as from its use as a biological weapon. The invention involves forming catechol-like functional groups.

Description

METHOD FOR SCREENING FOR TYPE B TRICHOTHECENE

MYCOTOXINS

Related Applications This application claims priority from U.S. Provisional Application Serial

Number 60/116,000 filed January 15, 1999, which is incorporated herein by reference.

Field of the Invention

This invention relates to a method for screening a sample for type B trichothecene mycotoxins and, more particularly, to electrochemical detection of type B trichothecene mycotoxins contamination from the natural growth of fungi such as in agricultural products as well as from its use as a biological weapon.

Background

Trichothecene mycotoxins (hereinafter referred to interchangeably as "trichothecenes," "trichothecene mycotoxins," or "mycotoxins") are metabolites produced by fungi such as Fusarium, Myrothecium, and Stachybotrys. The toxicological effects of trichothecene mycotoxins to microorganisms, plants, animals and humans include enteritis, emesis, dermonecrosis, gastroenteritis, oral necrosis and gastroenteric necrosis in livestock. Low-level effects in humans include nausea, vomiting, diarrhea and immunosuppression.

Contamination of agricultural products with trichothecene mycotoxins is a worldwide problem, threatening human health as well as the economy. In addition, trichothecene mycotoxins have been used as chemical weapons such as yellow rain used in Southeast Asia and Afghanistan in the late 70's. Recently, the United Nations Special Commission (UNSCOM) has suspected the Iraqi government of producing trichothecene mycotoxins as biological weaponry. The trichothecene mycotoxins of greatest concern as warfare agents include the type A trichothecene T-2 toxin, and the type B trichothecenes 4-deoxynivalenol (DON), diacetylnivalenol, and nivalenol which have lethal dose - 50 (LD₅₀) (intraperitoneal, in mice) of 5.2, 70.0, 9.6, and 4.0 mg/kg, respectively. 5 More than 80 different trichothecene mycotoxins have been isolated and identified from natural sources and have been grouped into types according to their chemical structure, where the common element consists of a 12,13-epoxytrichothec- 9-ene ring. Among the four types of trichothecene mycotoxins (types A, B, C and D), type A and type B (see Figure 1 ) are the most studied due to their toxicity and

10 frequent occurrences in agricultural products. As shown in Figure 1 , common type A trichothecene mycotoxins are verrucarol, scirpentriol, T-2 toxin, and iso-T-2-toxin. Common type B trichothecene mycotoxins are nivalenol (NIV), fusarenon-X (FX), deoxynivalenol (DON), 3-acetyl-DON, 15-acetyl-DON, and 3,15-diacetyl-DON.

A major portion of trichothecene mycotoxin contamination in agricultural

15 products consists of DON and nivalenol and their derivatives (type B) and to a lesser degree T-2 toxin and scirpentriol (type A). The concentration of type A trichothecene mycotoxins is correlated to the concentration of type B trichothecene mycotoxins; studies have shown that the concentration of type B trichothecene mycotoxins is typically two times higher than type A trichothecene mycotoxins (Parker, et al.,

20 1996).

Among these trichothecene mycotoxins, DON, commonly known as vomitoxin, is the most important and most studied due to its frequent occurrences in grains grown in North America. Due to the hazards of DON-contaminated grains, the United States Food and Drug Administration (FDA) has issued an advisory to

25 federal and state officials recommending that the concentration of DON not exceed 2.0 ppm (parts per million) in wheat entering the milling process, 1.0 ppm in finished wheat products for human consumption, and 4.0 ppm in animal feed ingredients.

Several analytical methods have been developed to detect these toxins. The most commonly used methods include gas chromatography (GC)/electron-capture

30 detector (ECD) methodology (Croteau, et al., 1994; Moller, et al., 1992; Laure, et al., 1991 ; Scott, et al., 1986; Cohen et al., 1984), thin layer chromatography (TLC)/mass spectroscopy (MS) methodology (Brumley, et al., 1985), TLC/Fluorescence (Trucksess, et al., 1987; Eppley, et al., 1986; Shannon, et al., 1985), high performance liquid chromatography (HPLC)/ultraviolet radiation (UV) methodology (Laure, et al., 1991 ), HPLC/MS (Kostiainen, et al., 1991 ; Voyksner, et al, 1987), and GC/MS methodology (Mossoba, et al., 1996; Krishnamurthy, et al., 1987; D'Agostino, et al., 1986). These methods require significant amounts of time associated with labor-intensive clean-up, sophisticated instrumentation, and skillful operators.

Recently, enzyme-linked immunosorbent assays (ELISA) have become more popular. Although ELISA may be ideal for detecting these toxins without extensive clean-up through the use of antibodies, their high degree of specificity limits their use to individual toxins; separate tests must be performed for each trichothecene mycotoxin. For example, to determine if type B trichothecene mycotoxins are present in a sample using ELISA, separate tests must be performed for NIV, FX, DON, 3-acetyl-DON, and 3,15-diacetyl-DON. ELISA cannot be used to screen generally for all type B trichothecene mycotoxins.

A rapid, simple and inexpensive method for type specific trichothecene mycotoxin detection that does not require extensive clean-up and sophisticated laboratory equipment would simplify and speed routine screening of trichothecene mycotoxins in food and animal feeds.

Electrochemical methods are ideal for these tasks because they require less intensive clean-up thereby allowing rapid and inexpensive detection. The recent development of chemical and biological sensors utilizing electrochemical detection schemes has demonstrated the feasibility of using electrochemical detection techniques. However, due to the difficulty in reducing or oxidizing trichothecene mycotoxins, electrochemical methods do not currently play a major role in their detection. A few studies have used electrochemical detectors (amperometric detection) combined with HPLC separation for detection of trichothecene mycotoxins where the applied potentials were -1.4 V or +1.0 V in basic solution. Two studies have explored the electrochemistry of trichothecene mycotoxins in detail (Visconti, et al., 1984; Palmisano, et al., 1981 ). In those studies, it was demonstrated that type A trichothecene mycotoxins are electrochemically inactive, while type B and C trichothecene mycotoxins could be reduced at potentials of approximately -1.4 V on

Hg electrodes versus saturated calomel electrode (SCE) on Hg electrodes.

However, at such extreme potentials electrochemical selectivity is compromised and extensive deoxygenation is required. Further, Visconti, et al. and Palmisano, et al. never identified the source of the electrochemical activity. Therefore, there exists a need to more easily reduce or oxidize type B trichothecene mycotoxins. Such a method would allow for easier type specific chemical or electrochemical detection of trichothecene mycotoxins and eliminate the need to separate test for each trichothecene mycotoxin. However, in order to develop such a method, the source of the electrochemical activity must be identified.

Summary The present invention is a simple and inexpensive method to chemically, or preferably electrochemically, detect type B trichothecene mycotoxins in grain samples, run-off, or other potentially contaminated samples. The present invention identifies the source of the electrochemical activity reported by Visconti, et al. and Palmisano, et al. and discloses that easily oxidizable or reducible compounds can be formed from type B trichothecene mycotoxins. These compounds are characterized by catechol-like functional groups with the following general form:

Because these compounds can be formed from all type B trichothecene mycotoxins, the method presented herein may be used to broadly screen for all type B trichothecene mycotoxin compounds, thereby eliminating the need to separately test for each one. In general, if compounds containing these catechol-like functional groups are detected in a sample, then type B trichothecene mycotoxins are present in the sample. Likewise, if these compounds are not detected in the sample, then type B trichothecene mycotoxins are absent from the sample.

Although compounds containing catechol-like functional groups may be present naturally, these groups can also be formed using laboratory conditions. Any method of forming these compounds containing catechol-like functional groups will be effective; however, the present invention discloses that the best mode is by hydrolysis. More specifically, type B trichothecene mycotoxins, such as DON and acetyl-DON, hydrolyze in basic solutions upon heating (Young, et al., 1986). Likewise any method of detecting these compounds will be effective. However, oxidation by electrochemical methods at potentials near +0.6 V will produce results at the detection limits required by the FDA. Based on the reaction pathway, the present invention can be extended to virtually all type B trichothecene mycotoxins because they have the same catechol-like functional group in the R7 and R15 positions as DON.

In a preferable embodiment of the present invention, type B trichothecene mycotoxins are detected in a sample by forming a compound with a catechol-like functional group and then detecting the presence of the compound. Preferably, the compound with a catechol-like functional group should be formed by hydrolyzing a sample in a basic solution of approximately 0.1 M NaOH at between about 75°C and 80°C for approximately one hour. Preferably, the hydrolysis products should be detected by initiating a redox reaction by electrochemical oxidization on glassy carbon electrodes or carbon fiber ultramicroelectrodes at potentials near +0.6 V. By correlating the peak current determined voltammetrically with the peak currents for oxidation of known catechol-like functional groups, the presence of type B trichothecene mycotoxins may be determined.

To enhance the results and obtain detection limits within that required by the FDA, the sample should be reacidified, preferably with approximately 1.0 M H₂SO₄, prior to electrochemical oxidation. Further, where solid samples are to be analyzed, such as grain or soil samples, extraction will enhance the results and improve detection limits. Preferably, extraction should be performed by grinding the sample and adding approximately 100 mL of an acetonitrile and water solution with a volume ratio of acetonitrile to water equal to approximately 85:15. The sample should then be vigorously stirred for approximately one hour and then filtered through a clean-up column (approximately 1.5 cm diameter) packed with a mixture containing about 5.0 grams of AI₂O₃ powder and about 0.15 grams of carbon powder. After filtration, the filtrate should be evaporated to dryness with a rotor evaporator at about 50°C.

In addition, a colorimetric test kit can be devised using an indicator, which changes color upon reduction, and with sufficient oxidizing power, e.g. KMnO_4ι to react with the hydrolysis products of the type B trichothecene mycotoxins. Such a kit would include a colorimetric testing means and an indicator with sufficient oxidizing power to oxidize catechol-like functional groups. The utility of this approach for the analysis of real-world samples was demonstrated using rice samples spiked with DON. Despite the fact that the data contained a sloping background and peak potential was slightly shifted, these results show that food products, such as rice extracts, will not introduce significant electrochemical interference and that DON can be quantified down to the 1 ppm level.

Accordingly, it is an object of this invention to provide a new and useful method to electrochemically detect trichothecene mycotoxins that does not require expensive laboratory equipment, skilled operators, and time consuming clean-up. A further object of this invention is to provide a method for type specific screening.

Yet another objective of this invention is to present a colorimetric test kit for type specific screening of trichothecene mycotoxins.

Further objects and advantages of this invention will become apparent upon consideration of the figures and related description.

Brief Description of the Drawings

Figure 1 is a diagram and table that depicts the structure of some common type A and type B trichothecene mycotoxins.

Figure 2 is a diagram that identifies the mechanism of DON hydrolysis in basic solution and its products.

Figure 3 is a graph that shows cyclic voltammograms equal to 1.2 mM 2-(2- hydroxyethoxy)phenol (dashed line) and hydrolysis products equal to 1.2 mM DON (solid line).

Figure 4 is a diagram that shows the proposed mechanism for the electrochemical reactions of norDON-B and norDON-C.

Figure 5 is a graph that represents square wave voltammograms of different concentrations (1.2 mM, 0.24 mM, 60 μM and 12 μM) of DON and the blank solution.

Figure 6 is a graph that depicts a calibration curve of DON (peak current versus concentration). Figure 7 is a graph that depict (a) square wave voltammograms of samples with 0, 1 , 3, 4, and 5 ppm of DON in 25 grams of rice samples (final extraction volume equal to 2.2 mL) and (b) background-subtracted square wave voltammograms of samples with 1 , 3, 4 and 5 ppm DON in rice samples (subtractions was performed by subtracting an estimated linear baseline beneath the peaks.

Figure 8 is a graph that depicts the calibration curve of DON in rice samples. The sensitivity is equal to 0.134 μA/ppm and the correlation coefficient is equal to 0.995.

Detailed Description

The present invention is a simple and inexpensive method for the detection of type B trichothecene mycotoxins. The present invention discloses that compounds with a catechol-like functional group can be formed from type B trichothecene mycotoxins and that these compounds can be easily oxidized or reduced.

When these compounds are formed by hydrolysis and the redox reaction is initiated using electrochemical techniques, a detection limit for DON of 9.1 μM in solution, corresponding to 0.24 ppm in a 25-gram grain sample where the final extraction volume is 2.2 mL, can be acheived. The linear dynamic detection range is from 0.32 ppm to greater than 32 ppm.

The following description of materials, instruments, electrodes, and methods used during experimentation is not intended to be limiting and is provided merely for reference; equivalent materials, instruments, electrodes, and methods can be employed with similar results. One skilled in the art would identify other methods of forming a compound with a catechol-like functional group from type B trichothecenes. Likewise, other methods of chemically and electrochemically detecting these compounds can be employed. Other methods may be employed to enhance the results obtained in the present invention. The methods disclosed, such as extracting the sample and reacidifying the sample prior to oxidation, are not intended to be limiting.

Experimental Procedure

DON, T-2 toxin, verrucarin A, and verrucarol were provided by Professor Bruce Jarvis in Department of Chemistry & Biochemistry, University of Maryland at

College Park, AI₂O₃ powder (neutral, 150 mesh) and 2-(2-hydroxyethoxy)phenol was purchased from Aldrich, and decolorizing carbon powder (Norit-A, alkaline) was obtained from Fisher. DON was used as a representative compound for type B trichothecene mycotoxins. All compounds were used as received without any further purification. Glassy carbon disk electrodes were used as working electrodes, a silver wire was used as a quasi-reference electrode, and a Pt foil was used as a counter electrode. Working electrodes were polished with 3 μm diamond paste then colloidal silica. After polishing, electrodes were ultrasonicated for at least three (3) minutes in distilled/deionized water.

A BAS 100A potentiostat was used for all electrochemical measurements. Cyclic voltammetry was performed from +200 mV to +800 mV with scan rates ranging from 5 to 500 mV/s. Waveform parameters for square-wave voltammetry (SWV) included: (1) step potential equal to 4 mV, (2) square-wave amplitude equal to 25 mV, and (3) square-wave frequency equal to 15 Hz. After each measurement, the solution was stirred to re-establish initial surface concentration of analyses.

The samples were prepared by heating a 3 mL aqueous solution of DON in approximately 0.1 M NaOH at between about 75°C to 80°C for approximately one hour to hydrolyze the sample. Hydrolysis is a function of time, temperature and pH. Therefore, the rate of hydrolysis can be manipulated by adjusting the pH and the temperature. To prevent evaporation, the solution was kept in a capped vial while heating. Then the sample was cooled to room temperature and acidified with about 2 mL of approximately 1 M H₂SO₄ to reacidify the sample prior to electrochemical oxidation to create better defined peak currents and improve the detection limit. Although NaOH and H₂SO₄ were used in this example, any equivalent base and acid may used to hydrolyze and oxidize the sample, respectively.

Extraction of DON from Rice Samples

Prior to analysis, a commercial sample of rice was spiked with DON by grinding the rice into a fine powder at 12,200 rpm in a mill for 1.5 minutes. A 25- gram portion of rice powder was then spiked with a 12.5 μg/mL DON/methanol stock solution. The spiked sample was allowed to dry. To enhance the results and improve the detection limit, about 100 mL of an approximately 85:15 (by volume) acetonitrile to water extraction solution was added to the dried sample. The sample was then vigorously stirred for one hour. The extract was filtered through a cleanup column (approximately 1.5 cm diameter) packed with a mixture containing about 5.0 grams of AI₂O₃ powder and about 0.15 grams of carbon powder. After filtration, 70 mL of the filtrate was evaporated to dryness with a rotor evaporator at about 50°C. The residue was redissolved in approximately 1.5 mL of about 0.1 M NaOH and heated at between about 75°C to 80°C for about one hour to hydrolyze the DON. After being cooled to room temperature, the extract was acidified with about 0.7 mL of approximately 1.0 M H₂S0₄to enhance the results. The final volume of the extract was 2.2 mL.

Electrochemical Properties of the Hydrolysis Products of DON DON and acetyl-DON in basic solutions at a temperature between about 75°C and 80°C degrade to at least four (4) products: norDON-A, norDON-B, norDON-C and lactone. The mechanism of forming norDON-A, B and C has been proposed by Young, et al. (1986) and is shown in Figure 2. The present invention illustrates that some of the hydrolysis products are responsible for electroactivity. Based on Young, et al.'s structural information, it was hypothesized that norDON-B and norDON-C were responsible for the electroactivity due to the catechol-like functional group on norDON-B and norDON-C. A model compound, 2- (2-hydroxyethoxy)phenol, whose structure is similar to the portion of norDON-B and norDON-C that is believed to be electroactive, was used to verify this hypothesis. Figure 3 shows the cyclic voltammograms of 2-(2-hydroxyethoxy)phenol and a hydrolyzed DON sample. For the purpose of comparison, the response of 2-(2- hydroxyethoxy)phenol was normalized to the same peak current obtained for the hydrolyzed DON sample. The redox wave of 2-(2-hydroxyethoxy)phenol was almost identical to that of the hydrolysis products of DON, suggesting that the hydrolysis products and 2-(2-hydroxyethoxy)phenol share the same electroactive moiety. The anodic peak potential of the hydrolyzed sample occurred at approximately 600 mV at pH equal to 0.5. The absence of a cathodic peak in the return scan suggested that the oxidation products of norDON-B and norDON-C rapidly converted to another electrochemically inactive species. The redox mechanism seemed to involve 2 e^", 1 H⁺ transfer followed by an irreversible chemical reaction with water, as shown in Figure 4, in a manner similar to that reported for tocopherol [need reference]. A subsequent ring-opening reaction was possible, resulting in quinone species; however, no redox peaks were observed at +0.2 V, as would be expected for a quinone species. Therefore, the ring-opening reaction did not occur or occurred slowly relative to cyclic voltammog ramie experiments.

The structural differences between norDON-B and norDON-C were too minor to exhibit any measurable differences in their electrochemical properties, as indicated by the single peak in Figure 3. Therefore, it was only possible to determine the conversion efficiency from DON to all electroactive products. By comparing the peak current of 1.2 mM hydrolyzed DON to the peak current of 1.2 μM 2-(2- hydroxyethoxy)phenol under identical conditions, the conversion efficiency was estimated to be approximately 18%. The redox reaction of the electroactive products produced diffusion-controlled responses with no indication of adsorption as indicated by a slope equal to 0.5 of a log-log plot of peak current versus scan rate.

The redox behavior of the hydrolyzed DON was very well behaved. The scan- to-scan reproducibility of the responses showed that the oxidation products did not foul the electrode surface, which is typically a concern for electrochemical methods. After scanning 35 times, the final cyclic voltammogram was almost identical to the first with only a slight decrease in peak height. Following an initial decrease in peak current of 2.1% in the first four (4) scans, the peak current decreased by only 1.4% over a subsequent 30 scans. Thus, it was concluded that surface fouling is minimal in the procedure presented.

Quantification of DON Through its Hydrolysis Products

Cyclic voltammetry is a powerful technique for the characterization of redox reactions. However, it is less sensitive for analytical applications due to its relatively large background. For calibration and quantification, square wave voltammetry (SWV) was used. Figure 5 shows the SWVs of various concentrations of DON in solution. Although glassy carbon electrodes were used for the data presented in this work, similar results can be obtained with carbon fiber ultramicroelectrodes.

Figure 6 shows a calibration curve for DON using the present invention. The number of measurements at each concentration was 12. The sensitivity for this method was determined to be 4.2 μA/mM. The limit of detection for DON, based on a 99% confidence interval, was determined to be 9.1 μM in solution. Based on these results, if a 25-gram sample was used and the total final extraction volume was 5 mL, the limit of detection would correspond to 0.24 ppm by weight in the sample. The linear dynamic range was determined to be from 12 μM to greater than 1200 μM, corresponding to a range of 0.32 ppm to greater than 32 ppm. The level of concern for DON, as established by the Food and Drug Administration (FDA), is 2.0 ppm DON in wheat entering the milling process, 1.0 ppm in finished wheat products for human consumption, and 4.0 ppm in animal feed ingredients. These results suggest that the detection range of this method is suitable for detecting trichothecene mycotoxins in typical agricultural samples.

The time required to obtain a maximum yield of the electroactive products under conditions of approximately 0.1 M NaOH and heating at between about 75°C to 80°C was approximately one hour. Ideally, a shorter hydrolysis time will make this method more appealing. Because the hydrolysis rate is a function of temperature and pH, the hydrolysis time may be shortened if a higher temperature and/or pH are used.

Quantification of DON in Spiked Rice Samples

Rice samples were used to demonstrate the utility of this method for the detection of DON in complex samples. Figure 7a shows the voltammograms of DON obtained from filtered rice extracts. The concentrations of DON spiked in the original rice samples were 0, 1 , 3, 4, and 5 ppm. The control sample (0 ppm) shows no redox peak on top of the sloping background. The spiked samples show similar sloping background currents with a visible oxidation peak at approximately 700 mV, and approximately 100 mV more positive than that observed in standard solutions. The sloping background and shifted peak potential are likely due to matrix effects in the rice extract but do not adversely influence the analysis using this method. Following background subtraction, symmetrical responses were obtained by extrapolating a linear background under the peaks as shown in Figure 7b. Figure 8 shows the calibration curve determined for DON in filtered rice extracts. The sensitivity derived from the calibration curve was 0.18 μA/ppm for a 25-gram sample with a 2.2 mL final extraction volume. The average peak current for 1 ppm DON was determined to be 0.102 μA (n=6) at a peak potential of 736 mV. The standard deviation of the background current for the blank rice extract at 736 mV was 0.009 μA (n=5). This results in a signal to noise ratio (S/N) blank for 1 ppm DON in the rice extract equal to 11 , which exceeds the level of quantification (S/N equal to 10). Therefore, it can be concluded that this method is useful for quantifying DON concentrations in rice samples to less than 1 ppm.

Type Specific Detection with the Indirect Electrochemical Method In the example presented, DON was used as a representative compound for the type B trichothecene mycotoxins. Young, et al. (1986) reported that acetyl-DON and di-acetyl-DON rapidly convert to DON, and tri-acetyl-DON hydrolyzes to isoDON in less than 30 seconds in basic solutions at a temperature of between about 75°C to 80°C. Consequently, DON and acetyl derivatives of DON have the same hydrolysis products.

Close examination of the hydrolysis reaction pathway suggests that the formation of the electroactive portion of the products only involves the R7 and R15 functional groups in DON. Common type B trichothecene mycotoxins such as DON, nivalenol, tri-acetyl-DON, di-acetyl-DON, acetyl-DON and fusarenon-X should produce similar electroactive products because, at some point during their hydrolysis, they all have the same R7 and R15 functional groups. As a result, this method can be used for type B specific screening. Because similar behavior was not observed with type A (T-2 and varracarol) or type C (cerrucarin-A) trichothecene mycotoxins, this method appears to be specific for type B trichothecene mycotoxins. The above-referenced methods utilize hydrolysis to form the compounds with a catechol-like functional group, however, hydrolysis is not the only method to form these compounds. Further, it is not necessary that these compounds be formed by hydrolysis; it is anticipated that any method that forms compounds with catechol-like functional groups from type B trichothecene mycotoxins will yield equivalent results. In addition, although the preceding description relies on electrochemical techniques are used to initiate the redox reaction and detect the presence of these compounds, other commonly known methods to initiate redox reactions will work, provided they have sufficient oxidizing power. For example, KMnO₄, a common oxidizing agent, was employed and supplied sufficient oxidizing power to detect the presence of the compound.

Further, colorimetric methods can be used to detect the presence of these trichothecene mycotoxins provided the indicator has sufficient oxidizing power to react with the hydrolysis products of the type B trichothecene mycotoxins.

Claims

CLAIMS What is claimed is:

1. A method for screening a sample for the presence of type B trichothecene mycotoxins, comprising the steps of: a. providing a sample; and, b. chemically detecting a compound with a catechol-like functional group in said sample wherein the existence of said compound indicates the presence of type B trichothecene mycotoxins and the nonexistence of said compound indicates the absence of type B trichothecene mycotoxins.

A method as in Claim 1 , wherein the step of chemically detecting a compound with a catechol-like functional group is comprised of initiating a redox reaction.

3. A method as in Claim 1 , wherein the step of chemically detecting a compound with a catechol-like functional group is comprised of electrochemically initiating a redox reaction.

4. A method as in Claim 3, wherein the step of electrochemically initiating a redox reaction is performed with glassy carbon electrodes.

5. A method as in Claim 3, wherein the step of electrochemically initiating a redox reaction is performed with carbon fiber ultramicroelectrodes.

6. A method as in Claim 1 , further comprising the step of forming a compound with a catechol-like function group in said sample prior to the step of chemically detecting said compound.

7. A method as in Claim 6, wherein said step of forming a compound with a catechol-like functional group is comprised of hydrolyzing the sample.

8. A method as in Claim 7, wherein said step of hydrolyzing said sample is comprised of heating said sample under basic conditions for a time period sufficient to hydrolyze a type B trichothecene mycotoxin.

9. A method as in Claim 8 wherein the step of heating said sample is performed at a temperature of between about 75° C to 80°C for approximately one hour in a NaOH solution.

5 10. A method as in Claim 9, wherein said NaOH solution has a concentration of approximately 0.1 M.

11. A method as in Claim 1 , further comprising the step of extracting the sample prior to chemically detecting a compound with a catechol-like functional group

10 in said sample.

12. A method as in Claim 11 , wherein the step of extracting the sample, is comprised of the following steps: a. grinding said sample;

15 b. adding an acetonitrile and water solution to said sample; c. stirring said sample; d. filtering said sample; and e. drying said sample.

20 13. A method as in Claim 12, wherein said solution has a volume ratio of acetonitrile to water equal to approximately 85:15.

14. A method as in Claim 12, wherein the step of stirring said sample occurs for approximately one hour.

25

15. A method as in Claim 12, wherein the step of filtering said sample is performed with a clean-up column.

16. A method as in Claim 15, wherein said clean-up column has a diameter of 30 approximately 1.5 cm and is packed with a mixture comprised of about 5.0 grams of AI₂O₃ powder and about 0.15 grams of carbon powder.

17. A method as in Claim 12, wherein the step of drying said sample is performed by evaporating said sample to dryness with a rotor evaporator at about 50°C.

18. A method as in Claim 1 , further comprising the step of acidifying the sample prior to chemically detecting a compound with a catechol-like functional group.

19. A method as in Claim 18, wherein the step of acidifying the sample is performed with a H₂SO₄ solution.

20. A method as in Claim 19, wherein said H₂SO₄ solution has a concentration of about 1.0 M.

21. A method as in Claim 1 , further comprising the step of correlating the detection results with known catechol-like functional groups.

22. A method as in Claim 21 , wherein said step of chemically detecting a compound with a catechol-like functional group is comprised of initiating a redox reaction and said step of correlating the detection results is comprised of: a. measuring the peak current of a compound with a catechol-like functional group; and b. comparing the measured peak current with a electrochemical peak current of a known catechol-like functional group.

23. A method as in Claim 22, wherein the peak current of a compound with a catechol-like functional group and the peak current of a known catechol-like function group are determined voltammetrically.

24. A method for screening a sample for the presence of type B trichothecene mycotoxins, comprising the steps of: a. providing a sample; b. grinding the sample; c. adding to said ground sample a solution of acetonitrile and water with a volume ratio of acetonitrile to water equal to about 85:15 d. stirring the sample for approximately one hour; e. filtering the sample with a clean-up column with a diameter of approximately 1.5 cm and packed with a mixture comprised of about 5.0 grams of AI₂O₃ powder and about 0.15 grams of carbon powder; f. evaporating said sample to dryness with a rotor evaporator at about 50°C; g. heating the sample at between about 75°C to 80°C for approximately one hour in approximately 0.1 M NaOH solution; h. acidifying the heated sample with approximately 1.0 M H₂SO₄ solution; i. subjecting the heated, acidified sample to electrochemical oxidization with glassy carbon electrodes; j. measuring the peak current of the sample; and k. correlating the peak current determined voltammetrically with a peak current of a known catechol-like functional group.

25. A method for screening a sample for the presence of type B trichothecene mycotoxins, comprising the steps of: a. providing a sample; b. grinding the sample; c. adding to said ground sample a solution of acetonitrile and water with a volume ratio of acetonitrile to water equal to about 85:15 d. stirring the sample for approximately one hour; e. filtering the sample with a clean-up column with a diameter of approximately 1.5 cm and packed with a mixture comprised of about 5.0 grams of AI₂O₃ powder and about 0.15 grams of carbon powder; f. evaporating said sample to dryness with a rotor evaporator at about 50°C; g. heating the sample at between about 75°C to 80°C for approximately one hour in approximately 0.1 M NaOH solution; h. acidifying the heated sample with approximately 1.0 M H₂SO₄ solution; i. subjecting the heated, acidified sample to electrochemical oxidization with carbon fiber ultramicroelectrodes; j. measuring the peak current of the sample; and k. correlating the peak current determined voltammetrically with a peak current of a known catechol-like functional group.