Preparation of Samples

Aflatoxin contamination of food and feeds is usually in the range of ngg-1 to |g g_1. Sampling error is a severe problem in aflatoxin determination because only a few affected kernels can contaminate a large amount of finished product. Amounts as high as 207 000ngg~1 have been found in individual corn kernels. This is sufficient aflatoxin to produce a level of contamination of 20 ngg-1 in a batch of 10 000 kernels of grain. Sampling plans have been developed for various commodities. In general, the larger the unit size of the commodity, the larger the sample size should be. The sample should be finely ground and mixed before taking out the analytical test portion. Often the sampling error is larger than the analytical error.

Various methods of analysis have been devised. Many of these have been published in the Official Methods of Analysis of AOAC International, after collaborative studies by several laboratories. If the precision and accuracy of the results are acceptable the method becomes official.

The three most widely used extraction and cleanup methods for preparing aflatoxin extracts for TLC are the CB method, the BF method and the im-munoaffinity column method. The CB method, named after the Contaminants Branch of the US Food and Drug Administration (FDA), uses chloroform extraction, filtering through paper, addition to a silica gel column, washing with hexane and ether, elution with chloroform-methanol (97 : 3 v/v), and evaporation to dryness to prepare the extract for TLC. The BF method, named after the Best Foods Company, uses methanol-water (55 : 45 v/v) extraction, hexane

Aflatoxin M

Figure 1 Structures of aflatoxins B1, B2, G1, G2 and M1.

Aflatoxin M

Figure 1 Structures of aflatoxins B1, B2, G1, G2 and M1.

defatting in a separatory funnel, partition into chloroform and evaporation to dryness to prepare the extract for TLC. The immunoaffinity column method uses methanol-water (7:3 v/v) for extraction, filtering through paper, dilution with water, filtering through a glass microfibre filter, application to a column upon which antibodies to aflatoxins have been bound, washing with water, elution with methanol and evaporation to dryness to prepare the extract for TLC.

The advantage of the CB method is that it is precise and accurate when correctly performed. Disadvantages include the acquisition and disposal costs of the reagents used. The advantage of the BF method is that it has the lowest cost of any of the methods. The disadvantage is that it results in a somewhat dirtier extract. The advantages of the immunoaffinity column method are its simplicity of performance and the purity of the aflatoxins in the extract. Its disadvantage is the high cost of the columns.

After evaporation, in all three methods, the extract is carefully transferred using rinses of chloroform to a small vial. The solvent is again evaporated to dry-ness in a water bath under a stream of nitrogen. The residue is dissolved in a small amount of solvent (200 |L), usually benzene-acetonitrile (98 : 2 v/v), for spotting on TLC. Since the use of benzene is sometimes prohibited because of its toxicity, other solvents such as toluene-acetonitrile (9 : 1 v/v) may be used as well.

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