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45 Mycotoxins Methods​

45-01.01 Sampling Grain for Mycotoxins

This method provides a realistic approach for sampling corn, small grains, oilseeds, and pulses for mycotoxins. Mycotoxin contamination of cereal grains is likely to occur in pockets of high concentration, randomly distributed. Levels as high as 100,000 ppb have been reported in a single corn kernel. The U.S. Food and Drug Administration (FDA) has determined that the action level of mycotoxin is 20 ppb. One infected kernel of corn (with 100,000 ppb aflatoxin) may bring a sample of 5000 kernels (about 1500 g) to the FDA action level. Therefore, to try to make each 1.5-g sample representative, a contaminated seed should be reduced to about 1000 pieces. For this reason, the sampling and sample preparation guidelines must be carefully followed.

45-05.01 Aflatoxin—Thin-Layer Chromatography Method

This method determines the concentration of aflatoxin by thin-layer chromatography (TLC) of extracts of corn and soybeans. Aflatoxins are extremely potent carcinogens to a number of animals and should be regarded as potentially hazardous to humans. Caution: whenever possible, perform operations under a hood and use a glove box when toxins are in a dry state because of their electrostatic nature. Use gloves and a mask for personal protection when handling toxin or highly contaminated commodities. Swab accidental spills with 5% sodium hypochlorite bleach and rinse glassware with bleach before washing to decontaminate. See Note 1.

45-14.01 Aflatoxin—Rapid Screening Method

This method detects total aflatoxin (>=10 µg/kg, or ppb) by minicolumn chromatography of extracts of yellow and white corn and raw peanuts. The method can be used outside of the laboratory. See Note 1.

45-15.01 Aflatoxin—Presumptive Test

The bright greenish yellow fluorescence (BGYF) test (black light test) is used as a presumptive test to identify lots of white and yellow corn that may contain aflatoxins. Corn is inspected under ultraviolet (UV) light (365 nm) for the characteristic BGYF associated with Aspergillus flavus or A. parasiticus, the fungi that produce aflatoxins (Refs. 3, 4). If BGYF is present, indicating a positive test, corn must be further analyzed by thin-layer chromatographic, high-performance liquid chromatographic, or immunoassay methods to determine the presence and levels of aflatoxins. False negative tests rarely appear.

Growth of these fungi may or may not result in aflatoxin production. BGYF is also produced on grain sorghum or cottonseed, but usually not on seed killed by high-temperature drying. Aflatoxins do not fluoresce bright greenish yellow under long-wave UV light, but BGYF may be produced by reaction of kojic acid formed by fungi and peroxidase enzyme from the seed. BGYF test is the quickest method of detecting grain that could contain aflatoxin, requires very little equipment, and can easily be done in the field. However, a positive test does not absolutely indicate that aflatoxin is present. It identifies corn lots that should be tested for aflatoxin.

45-16.01 Aflatoxin—Liquid Chromatography Method

Aflatoxins are extracted and purified, derivatized with trifluoroacetic acid (TFA) (aflatoxins B₁ and G₁ to B_2a and G_2a, respectively), separated by reversed-phase high-performance liquid chromatography (HPLC), and detected by fluorescence. The method can measure 0.1 ng of aflatoxins B₁, B₂, G₁, and G₂. The detection limit is about 0.3 ng/g for each aflatoxin. This method is applicable to determination of aflatoxins B₁, B₂, G₁, and G₂ at >=13 ng total aflatoxin per g or 5 ng aflatoxin B₁ per g in corn and peanut butter​

45-20.01 Zearalenone—Thin-Layer Chromatography Method

This method determines concentration of zearalenone in corn by thin-layer chromatography (TLC).

45-21.01 Zearalenone and alpha-Zearalenol in Corn—HPLC Method

This method detects zearalenone and alpha-zearalenol (>=50 (µg/kg) by liquid chromatography of extracts of corn.

45-25.01 Identification of Aflatoxin on TLC Plates

This method identifies aflatoxins B₁ and G₁ in partially purified extracts of grains, nuts, oilseeds, and other agricultural commodities by formation of water-adduct derivatives (aflatoxins B_2a and G_2a) on thin-layer chromatography (TLC) plates. Although their effects on humans are not known at present, aflatoxins are extremely potent carcinogens to a number of animals and should be regarded as potentially hazardous to humans. Whenever possible, perform operations under a hood and use a glove box when toxins are in a dry state because of their electrostatic nature. Use gloves and a mask for personal protection when handling toxin or highly contaminated commodities. Swab accidental spills with 5% sodium hypochlorite bleach and rinse glassware with bleach before washing to decontaminate.

45-41.01 Deoxynivalenol in Wheat—TLC Method

This method detects concentration of deoxynivalenol (DON) at >300 ng/g by thin-layer chromatography (TLC) of extracts of wheat.

45-51.01 Fumonisins B₁, B₂, and B₃ in Corn—Liquid Chromatographic Method

Fumonisins are hepatotoxic and carcinogenic to rats, but their effects on humans are not fully known. This method is applicable for determination of fumonisins B₁, B₂, and B₃ in corn at > 1 µg/g. See Note 1.