Sampling and analysis

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The control of the mycotoxin problem comprises (a) the identification of the nature and extent of the problem (by the implementation of surveillance studies), (b) the introduction of improved handling procedures, which address the identified problems, and (c) the regular monitoring of foods and feeds as part of a quality control programme.

The operation of both surveillance studies and quality control programmes requires efficient sampling and analysis methods.

Since the distribution of aflatoxins (and, presumably, other mycotoxins) in grains is highly skewed, it is important that great care is taken to collect a representative sample (Coker and Jones, 1988). There is still considerable debate as to the appropriate size of such samples. In general, the sample size should increase with increasing particle size; samples of whole groundnuts, maize and rice, for example, should be of the order of 20, 10 and 5kg respectively. Samples of oilseed cake and meal should be approximately 10kg in weight. For whole grains, each sample should be composed of about 100 incremental samples, collected sytematically from throughout the batch, whereas samples of cake and meal require approximately 50 increments. It is important to remember that the collection of samples from the surface of a large, mature stack of grains will only reflect the quality of the outer layers. The mycotoxin content of the grain in the interior of the stack can only be monitored during the break-down of the stack. Needless to say, an incorrectly collected sample will invalidate the final analysis result.

The sampling of grain shipments, normally involving tens of thousands of tonnes of material, poses a particularly difficult sampling problem. Representative samples should be collected from carefully defined 500 tonne batches, using the methods described above. Potential sampling points include weighing towers, conveyor belts, and trucks and barges receiving the discharged material. The sampling of fast moving grain is a hazardous operation; automatic, on-line sampling equipment should be used wherever possible.

The reduction of the sample, for analysis, should also be performed so as to ensure the representative nature of the laboratory sample. It is imperative that the complete sample is comminuted prior to subdivision. Ideally, the comminution and subdivision of whole grains should be performed simultaneously, using a subsampling mill. Alternatively, the comminuted sample should be subdivided using a mechanical riffle. Manual coning and quartering procedures should only be used as a last resort.

Equipment available for the collection of representative samples is discussed in detail in Chapter 3.

High performance liquid chromatography (HPLC) has been used for the analysis of a wide range of mycotoxins including the aflatoxins, ochratoxin A, zearalenone, deoxynivalenol (DON) and the fumonisins. To date, high performance thin layer chromatography (HPTLC) has been applied mainly to the aflatoxins whereas gas liquid chromatography (GLC) has been utilised for the quantification of DON, T-2 toxin and zearalenone. Enzyme-linked immunosorbent assays (ELISA) have also been applied to many mycotoxins including the aflatoxins, ochratoxin A, deoxynivalenol, T-2 toxin and zearalenone. Despite the utilisation of sophisticated, expensive HPLC, HPTLC, GLC and ELISA procedures, agreement between laboratories is invariably poor, when identical samples are analysed (Coker, 1984)!

Quality control programmes require simple, rapid, efficient analysis methods which can be handled by relatively unskilled operators (Coker, 1991). Recently developed rapid methods include those that utilise immunochemistry technology or selective adsorption agents. A rapid ELISA method for estimating aflatoxin in groundnuts, cottonseed, maize, rice and mixed feeds has been subjected to a collaborative study and recommended for First Action Approval by the Association of Official Analytical Chemists (AOAC). Solid phase ELISA kits have been developed for the aflatoxins, ochratoxin A, zearalenone and T-2 toxin in a variety of commodities. An 'immunodot' cup test, where the antibody is immobilised on a disk in the centre of a small plastic cup, has been approved by the AOAC as an Official First Action screen for aflatoxin in groundnuts, maize and cottonseed. Card tests have also been developed where the antibody is immobilised within a small indentation on a card similar in size to a credit card. Such tests have been developed for the aflatoxins, ochratoxin A, T-2 toxin and zearalenone in maize. The reported analysis (extraction, filtration and estimation) time for solid phase ELISA kits is 5-10 minutes. ELISA kits, however, are relatively expensive and suffer reduced shelf-lives at elevated temperatures.

Minicolumns (small glass columns) containing selective adsorption agents have been developed for aflatoxin/zearalenone (single test) and deoxynivalenol.

There is an urgent need for simple, robust, low-cost analysis methods, for the major mycotoxins, which can be routinely used in developing country laboratories.

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