Toxins : Aflatoxins
Aflatoxins are toxic metabolites produced by certain fungi in/on foods and feeds. They are probably the best known and most intensively researched mycotoxins in the world. Aflatoxins have been associated with various diseases, such as aflatoxicosis, in livestock, domestic animals and humans throughout the world. The occurence of aflatoxins is influenced by certain environmental factors; hence the extent of contamination will vary with geographic location, agricultural and agronomic practices, and the susceptibility of commodities to fungal invasion during preharvest, storage, and/or processing periods. Aflatoxins have received greater attention than any other mycotoxins because of their demonstrated potent carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans. As it is realized that absolute safety is never achieved, many countries have attempted to limit exposure to aflatoxins by imposing regulatory limits on commodities intended for use as food and feed.
In Raw Agricultural Products:
Aflatoxins often occur in crops in the field prior to harvest. Postharvest contamination can occur if crop drying is delayed and during storage of the crop if water is allowed to exceed critical values for the mold growth. Insect or rodent infestations facilitate mold invasion of some stored commodities.
Aflatoxins are detected occasionally in milk, cheese, corn, peanuts, cottonseed, nuts, almonds, figs, spices, and a variety of other foods and feeds. Milk, eggs, and meat products are sometimes contaminated because of the animal consumption of aflatoxin-contaminated feed. However, the commodities with the highest risk of aflatoxin contamination are corn, peanuts, and cottonseed.
In Processed Foods:
Corn is probably the commodity of greatest worldwide concern, because it is grown in climates that are likely to have perennial contamination with aflatoxins and corn is the staple food of many countries. However, procedures used in the processing of corn help to reduce contamination of the resulting food product. This is because although aflatoxins are stable to moderately stable in most food processes, they are unstable in processes such as those used in making tortillas that employ alkaline conditions or oxidizing steps. Aflatoxin-contaminated corn and cottonseed meal in dairy rations have resulted in aflatoxin M1 contaminated milk and milk products, including non-fat dry milk, cheese, and yogurt.
Corn can be used to produce flour and starch products and this links back to the problem statement such as aflatoxins is a likely toxin to be found in foods produced by the company in the problem statement.
Recent Methods of Analysis for Aflatoxins in Foods and Feeds
Sampling and Sample Preparation:
Sampling and sample preparation remain a considerable source of error in the analytical identification of aflatoxins. Thus, systematic approaches to sampling, sample preparation, and analysis are absolutely necessary to determine aflatoxins at the parts-per-billion level. In this regard, specific plans have been developed and tested rigorously for some commodities such as corn, peanuts, and tree nuts; sampling plans for some other commodities have been modeled after them. A common feature of all sampling plans is that the entire primary sample must be ground and mixed so that the analytical test portion has the same concentration of toxin as the original sample.
All analytical procedures include three steps: extraction, purification, and determination. The most significant recent improvement in the purification step is the use of solid-phase extraction.
Test extracts are cleaned up before instrumental analysis(thin layer or liquid chromatography) to remove coextracted materials that often interfere with the determination of target analytes.
Thin layer chromatography (TLC), also known as flat bed chromatography or planar chromatography is one of the most widely used separation techniques in aflatoxin analysis. Since 1990, it has been considered the AOAC official method and the method of choice to identify and quantitate aflatoxins at levels as low as 1 ng/g. The TLC method is also used to verify findings by newer, more rapid techniques.
Liquid chromatography (LC) is similar to TLC in many respects, including analyte application, stationary phase, and mobile phase. Liguid chromatography and TLC complement each other. For an analyst to use TLC for preliminary work to optimize LC separation conditions is not unusual.
Liquid chromatography methods for the determination of aflatoxins in foods include normal-phase LC (NPLC), reversed-phase LC (RPLC) with pre- or before-column derivatization (BCD), RPLC followed by postcolumn derivatization (PCD), and RPLC with electrochemical detection.
Thin layer chromatography and LC methods for determining aflatoxins in food are laborious and time consuming. Often, these techniques require knowledge and experience of chromatographic techniques to solve sepatation and and interference problems. Through advances in biotechnology, highly specific antibody-based tests are now commercially available that can identify and measure aflatoxins in food in less than 10 minutes. These tests are based on the affinities of the monoclonal or polyclonal antibodies for aflatoxins. The three types of immunochemical methods are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunoaffinity column assay (ICA).
These are mostly chemical methods of detection but still provide an insight into the immunochemical methods such as ELISA and RIA which can used to detect aflatoxins in foods, such as flour and starch products produced by the company.
Confirmation of Identities of the Aflatoxins:
Although analytical methods might consist of different extraction, clean-up, and quantitation steps, the results of the analyses by such methods should be similar when the methods are applied properly. Since the reliability of the quantitative data is not in question, the problem still to be solved is the confirmation of identity of the aflatoxins. The confirmation techniques used involve either chemical derivatization or mass spectrometry (MS).
Monitoring Techniques for Assessing Human Exposure to Aflatoxins
In the last few years, new technologies have been developed that more accurately monitor individual exposures to aflatoxins. Particular attention has been paid to the analysis of aflatoxin DNA adducts and albumin adducts as surrogates for genotoxicity in people. Autrup et al.(1983) pioneered the use of synchronous fluorescence spectroscopy for the measurement of aflatoxin DNA adducts in urine. Urine samples collected after exposure to alfatoxins were found to contain 2,3-dihydroxy-2-(N7-guanyl)-3-hydroxyaflatoxin B1, trivially known as AFB-Gual. Wild et al.(1986) used highly sensitive immunoassays to quantitate aflatoxins in human body fluids. An enzyme linked immunosorbent assay (ELISA) was used to quantitate aflatoxin B1 over the range of 0.01 ng /ml to 10 ng/ml, and was validated in human urine samples. Using this method, aflatoxin-DNA adduct excretion into urine was found to be positively correlated with dietary intake, and the major aflatoxin B1-DNA adduct excreted in urine was shown to be an appropriate dosimeter for monitoring aflatoxin dietary exposure.