Aflatoxin analysis

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. 

Fig. 2 Postcolumn derivatization scheme for aflatoxin analysis 1, mobile phase 2, HPLC pump 3, injection valve 4, precolumn 5. analytical column 6, derivatizing agent solution 7, auxiliary HPLC pump 8, T-valve 9, oil or water bath 10, reaction coil 11, fluorescence detector 12, waste 13, chromatographic data handling system.

Food and Agriculture Organization of the United Nations, 1993. Sampling plans for aflatoxin analysis in peanuts and com. Report of a FAO technical consultation. Rome, Italy. 

Table 11.6 Overview on the different CRMs for aflatoxin analysis and corresponding certified values (adapted from Boenke, 1995)...

Aflatoxin Analysis. Immimoassays were performed at the various locations where samples were collected and repeated approximately 24 hours later in the laboratoiy (Food Toxicology Research Laboratory, Department of Nutrition and Food Science, University of Arizona). The four commercially available (US market) test kits, listed below, were used ... 

Goto, T. Manabe, M. Application of ELISA to aflatoxin analysis Results of preliminary tests of analysis kits on the market Rep. Natl. Food Res. Inst, 52 53-9. 1988. Japanese. 

Institute of Standards and Industrial Research of Iran (2000) Code of practice for sampling of pistachio and pistachio kernel for aflatoxin analysis. Islamic Republic of Iran, Institute of Standards and Industrial Research of Iran (ISIRI 5197). 

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. 

ELISA methods for aflatoxins assay have been available for more than a decade. The technology is based on the ability of a specific antibody to distinguish the three-dimensional structure of specific aflatoxins. The direct competitive ELISA is commonly used in aflatoxin analysis [74],... 

FIGURE 11.3 Principle of competitive ELISA for aflatoxin analysis, (a) Sample mixed with conjugate (b) mixed content added to antibody-coated well (c) aflatoxin binds to antibody in the first incubation (d) unbound materials are rinsed away in the washing step (e) substrate is added to develop color (f) stop solution is added to stop the reaction. (Adapted from Zheng, M. Z., Richard, J. L., and Binder, J. 2006. Mycopathologia 161 261-273. With permission.)... 

The introduction of mass spectrometry and the subsequent coupling of liquid chromatography to this very efficient system of detection has resulted in the development of many LC-MS or LC-MS/MS methods for aflatoxin analysis. Because of the advantages of specificity and selectivity, chromatographic methods coupled to mass spectrometry continue to be developed they improve detection limits and are able to identify molecules by means of mass spectral fragmentation patterns. 

Examples for Aflatoxins Analysis in Eood Using UPLC/MS... 

Rubert et al. [118] also compared different procedures for Barley sample pretreatment for aflatoxins analysis via UPLC-MS/MS. 

In conclusion, a broad range of detection techniques used for practical analysis and detection of aflatoxins are available. This chapter highlighted some recent developments and new techniques about aflatoxins analysis in food via UPLC/MS. As shown, though there have been several recent successes in detection of aflatoxins, new methods are still required to achieve higher sensitivity and address other challenges that are posed by these toxins. 

There are many methods for aflatoxin analysis. The CB and BF methods (AOAC, Secs. 26.026 - 26.036) for the determination of aflatoxin in grains are widely used however, they require large amounts of solvent and are time consuming. Trucksess et al. (1984) developed a simple, rapid, solvent-efficient method for determinating aflatoxin in corn and peanut butter using a disposable silica gel column. This method was used to analyze some of the Venezuelan corn and corn products. 

Post-column in-line photochemical derivatization permits fluorescence detection of the common aflatoxins Bl, B2, Gl, and G2 (60). Chromatographic evidence indicates that photolysis causes the hydration of the nonfluorescent Bl and Gl components to B2a and G2a components, respectively. Analysis of naturally contaminated com samples show no interfering peaks and permits the deterrnination of 1 and 0.25 ppb for Bl and B2, respectively. 

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. 

The well characterized reactions of carcinogens such as benzols] pyrene and aflatoxin B with DNA (39-50) suggested to us that an analysis of the kinds of mutations these agents induced could shed light on the contribution of specific DNA lesions to mutagenesis. 

Danley JM, Staggers S, Varner A, Llewellyn GC, Dashek WV. A combined biochemical and ultrastructural analysis of aflatoxin action on the endomembrane system of plant cells. Actual Bot 1982 129 5-13. 

Where specific Regulations are in force then the methods associated with the Regulations shall be accredited if the control laboratory wishes to offer enforcement of the Regulations to customers. Examples of these are methods of analysis for aflatoxins and methods of analysis for specific and overall migration for food contact materials. 

It turned out that meeting a 20 ppb limit was not excessively burdensome on major manufacturers of peanut butter and other peanut products, at least in the United States aflatoxin tended to concentrate in discolored or otherwise irregular peanuts, which, fortunately, could be picked up and rejected by modern electronic sorting machines. Manufacturers did, however, have to institute substantial additional quality control procedures to meet FDA limits, and many smaller manufacturers had trouble meeting a 20 ppb limit. An extensive USDA program of sampling and analysis of raw peanuts, which continues to this day, was also put into place as the first line of attack on the problem. 

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. 

Such analytical measurements are necessary to establish concentrations for most agents in the environment. How much benzene is present in the air at gasoline stations as a result of its evaporation from gasoline What is the concentration of arsenic in water running off the surface of a hazardous waste site where unknown amounts of arsenic were buried over many years What is the polychlorinated biphenyl (PCB) concentration in fish swimming in waters next to a hazardous waste site known to contain this substance How much aflatoxin is in a batch of peanut butter The most reliable answers to these questions are those resulting from chemical analysis. 

A second factor concerns the purity of the diet and water received by the animals. Careful chemical analysis is needed to ensure the absence of significant amounts of highly toxic chemicals, such as aflatoxin, metals such as lead, arsenic, or cadmium, or certain pesticides, that may be present in water and various feed ingredients. 

HPLC analysis of polycyclic aromatic hydrocarbons (PAH) in drinking water is one of the current and classical applications of fluorescence. In this case, the detector contains a fluorescence flow cell placed after the chromatographic column. This mode of detection is specifically adapted to obtain threshold measurements imposed by legislation. The same process allows the measurement of aflatoxins (Fig. 12.11) and many other organic compounds (such as adrenaline, quinine, steroids and vitamins). 

Figure 12.11—Comparison of UV andfluorescence detection after chromatographic separation. Aflatoxins, which are carcinogenic contaminants present in certain batches of grain cereals, are controlled by HPLC analysis. It can be seen that the peak intensities in UV detection vary with concentration whereas fluorescence detection is much more sensitive to aflatoxin G2 and B2. (Reproduced by permission of SUPELCO.)...

This methodology traditionally used by biologists is now applied in the areas of environment and food analysis (with pesticides, aflatoxins, anabolic steroids, PAHs). However, it is only possible to measure a compound if the adapted antibodies and enzyme conjugates are available. 

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