Aflatoxins from corn, peanuts

Extraction of Aflatoxins from Corn, Peanuts, and Peanut Butter... 

Elute column with 6 mL of chloroform/acetone (9 1). Bakerbond Application Note FF-013 Extraction of Aflatoxins from Corn, Peanuts, and Peanut Butter (See Suggested Reading, Chapter 1). 

Aflatoxins are potent carcinogenic, mutagenic and teratogenic metabolites produced by molds. The major food affected with aflatoxins are corn, peanuts, rice, cottonseeds, dried fruit and milk from ingestion (103). The US action standards established by FDA are 20 pg/Kg for foods consumed by humans and 0.5 pg/kg for milk. In the case of animal feed, the levels are from 100 to 300 pg/kg. Therefore, assays capable of detecting at these levels have to be developed, (see Table 1 (104,105)). Detection of aflatoxins entails conjugation of these small molecules with carrier proteins like bovine serum albumin to produce antibodies (20). A number of commercial kits for aflatoxins are available (see sections on kits and immunoaflinity purification). 

The extraction of aflatoxins (Fig. 5.9) from corn, peanuts, and peanut butter involves the dissolution of the aflatoxins in an aqueous methanol extract, followed by extraction with chloroform and clean-up on a silica-gel column. The extraction is carried out with 50 g of food material and 200 mL of methanol/water (85 15) for 30 min. Filter 40 mL of the extract and collect the filtrate. Add 40 mL of 10% sodium chloride to salt out the aflatoxin and extract with 2 X 25 mL of chloroform. Evaporate to dryness and redissolve with 3 mL of methylene chloride. Sample is ready for addition to the silica-gel column that contains 0.5 g of packing material. The mechanism of sorption involves hydrogen bonding between the oxygen atoms of the aflatoxin and the silica gel. Sorption is from methylene chloride and elution is carried out with chloroform/acetone (9 1). 

Figure 5.9. Structure of aflatoxins extracted from corn and peanuts.

Method. The sample is spotted on to a TLC plate (silica gel) followed by 2 d of a solution of TFA (50% in chloroform, prepared fresh each day). The reaction is allowed to proceed for 5 min, after which time the plate is dried with a stream of nitrogen or air. The plate is then developed with chloroform—acetone (in various ratios such as 9 1 or 4 1), benzene-ethanol-water (43 35 19) or ethyl acetate-methanol-water (96 3 1). The plate is removed from the tank and dried in the dark. The spots are observed visually under UV light at long wavelength. The derivative of aflatoxin Bt appears blue, while the derivative of aflatoxin Gi is green. This confirmation technique has been applied to peanuts, brazil nuts, pistachio nuts, walnuts, barley, corn and other foods. 

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