Aflatoxins, determination

Tests for mycotoxin contamination can be accomplished both on the finished product and on the raw form. The latter case prevents the manufacture of an unfit product, but it often implies trouble in the evaluation of the contamination in a batch. In this case, for a defined sample size, sample preparation, and analytical method, principles are available to evaluate the accuracy of the aflatoxin determination, depending on the availability of an accurate estimate of the variability associated with each step of the analytical sequence (11,12). A valuable effort to estimate the uncertainty of the analytical sequence as a whole was carried out by the FAO (13). 

A specific test mixture may be prepared for working in a special area, e.g. an aflatoxin mixture for aflatoxin determination. (However, a test mixture for the determination of the plate number should not include any peptides or proteins. These compounds need to be eluted by gradient, see Figure 18.7, whereas the plate number is calculated from an isocratic chromatogram.) In all other cases, a test mixture that satisfies the following criteria is needed ... 

Table 1 Performance characteristics for aflatoxin determination using LC with fluorimetric detection...

However, HPTLC and HPLC techniques complanent each other the HPTLC for preliminary work to optimize LC separation conditions during the development of a method or it may also use as screening for the analysis of a large number of samples to limit the HPLC analysis only to positive samples. Liquid chromatographic methods for aflatoxin determination include both normal and reverse-phase separations, although current methods for aflatoxin analysis typically rely upon reverse-phase HPLC, with mixtures of methanol, water, and acetonitrile for mobile phases. 

In Table 11.4, some analytical methods for aflatoxin determination have been included with their performance characteristics. 

Some Analytical Methods for Aflatoxins Determination and Their Performance Characteristics... 

Alcaide-Mohna, M., Ruiz-Jimenez, J., Mata-Granados, J. M., and Luque de Castro, M.D. 2009. High through-put aflatoxin determination in plant material by automated solid-phase extraction on-line coupled to laser-induced fluorescence screening and determination by hquid chromatography-triple quadrupole mass spectrometry. J. Chromatogr. A 1216 1115-1125. 

Holak, W DiProssimo, V. and Malek, E.G. (1997) Reductive voltanmietric hplc detection of aflatoxins determination of aflatoxin B1 in foods. J. Liq. Chromatogr. Related Technol, 20, 1057-1065. 

A. Farjam, N. C. van de Merbel, H. Lingeman, R. W. Frei and U. A. Th Brinkman, Non-selective desorption of immuno precolumns coupled on-line with column liquid chr O-matography determination of aflatoxins , Int. J. Environ. Anal. Chem. 45 73-87(1991). 

The two examples of sample preparation of solids containing low concentrations of the substances of interest will be the analysis of aflatoxins in corn meal (7) and the determination of the fungicide thiabendazole in citrus fruits. It should be pointed out that the applications chosen in this section attempt to reflect a range of analyses that the analyst is likely to meet in both research and industrial laboratories. 

Sharman, M. and Gilbert, J., Automated aflatoxin analysis of foods and animal feeds using immunoaffinity column clean-up and high-performance liquid chromatographic determination, /. Chromatogr., 543, 220, 1991. 

It is of interest to compare the shapes and sizes of various molecules with appreciable carcinogenic activity. For example, aflatoxin B] (VI), found in moldy peanuts and grain, is one of the most powerful carcinogens known. Its crystal structure was determined by van Soest and Peerdeman (46-48). 

A protocol approved by the FDA to determine the safety of low gossypol cottonseed kernels for human consumption was the basis for the second study by Reber (7 ). To prepare raw cottonseed flour, raw kernels were ground to meet Ro-tap sieve specifications of lab chow. To prepare roasted cottonseed flour, raw kernels were dry roasted at not less than 121°C for not less than 5 min. To prepare cooked cottonseed flour, raw kernels were cooked in steam until batch temperature had been at or above 121°C for 5 min. All cottonseed kernels were ground in the manner described above. The kernels contained not more than 0.037% (370 ppm) of free gossypol. They were free of Salmonella and did not contain detectable amounts of aflatoxin. The proximate analyses of the cottonseed flours are shown in Table I. 

An example of the importance of sampling is in the determination of aflatoxins (a class of highly toxic compounds... 

Other preharvest techniques include the development of more resistant crops (e.g., Becker, 1999 Guo et al., 1998), and biological control with atoxigenic A. flavus strains (Cotty, 1990 Dorner et al., 1998). Atoxigenic A. jiavus competed successfully with a toxic isolate when they were grown in mixed culture, obtaining a reduction of the aflatoxin content by 82 to 100%. This is a worthwhile approach for control, however possible side effects caused by a preemptive application of A. jiavus in the environment must be studied to determine potential risks to human and animal health. 

Did this FDA position make any scientific sense It implied that if aflatoxin could be detected by reliable analysis, it was too risky to be consumed by humans, but that if the aflatoxin happened to be present below the minimum detectable concentration it was acceptable. (Analytical chemists can never declare that a chemical is not present. The best that can be done is to show that it is not present above some level - 20 ppb in the case of aflatoxins, and other, widely varying, levels in the case of other chemicals in the environment.) To be fair to the FDA, perhaps the word acceptable should be withdrawn the agency s position was not so much that all concentrations of aflatoxin up to 20 ppb were acceptable, but that nothing much could be done about them, because the chemists could not determine whether they were truly present in a given lot of food until the concentration exceeded 20 ppb. 

Previous studies have determined that aflatoxins are biosynthesized by the polyketide metabolic pathway and are considered secondary metabolites b ause... 

Initial attempts to purify enzymes from A, parasiticus mycelial extracts that catalyze aflatoxin synthesis were unsuccessful because these enzymes are present in relatively low concentrations and are extremely short-lived (23). Subsequently, several techiuques were examined for disruption of large quantities of mycelia to obtain active and stable cell-fiee preparations (23, 24) pertinent enzymes were recovered from cell-free extracts after grinding mycelia under liquid nitrogen (24). The optimum age of mycelial cultures for recovery of aflatoxin pathway enzymes was determined to be between 72 and 84 h (24a, 25). 

The determination of trace components (halogens, heavy metals, pesticides, aflatoxins, and restsolvents) in flavor and fragrance materials used in foods and cosmetics is becoming increasingly important. 

Fig. 8.6 Estimated risk of liver cancer, P(d), in relation to dose of aflatoxin, d, as determined with different dose-incidence models. The models for the different curves. are as follows OH. one-hit model MS, multi-stage model W, Weibull model MH, multihit model MB, Mantel-Bryan (log-probit model) (from Krewski and Van Ryzin, 1981).

Figure 6.55 Log-log plot of relative carcinogenic potencies for a group of chemicals against mutagenic potency as determined by the Ames test. The potency is the amount of chemical giving tumors in 50% of the animals or 100 mutant colonies (revertants) of Salmonella bacteria. Aflatoxin B, is the most potent and benzidine the least potent. Abbreviations Moca, 4,4,-methylene-b/s-(2-chloroaniline) mms, methylmethane sulfonate. Source From Ref. 16.

Despite the fact that biological and predominantly immunoenzymatic methods have received increasing attention during the last decade for the determination of aflatoxins, chemical and immunochemical assays have to be preferred for their characteristics, including a lower limit of detection and high specificity. 

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