Aflatoxins identification

Because of its higher separation power, higher sensitivity, and accuracy, and the possibility of automating the instrumental analysis, HPLC is now the most commonly used technique in analytical laboratories. HPLC using fluorescence detection has already become the most accepted chromatographic method for the determination of aflatoxins. For its specificity in the case of molecules that exhibit fluorescence. Commission Decision 2002/657/EC, concerning the performance of analytical methods, considers the HPLC technique coupled with fluorescence detector a suitable confirmatory method for aflatoxin identification. 

Shier, W.T. et al.. Yellow pigments used in rapid identification of aflatoxin-producing Aspergillus strains are anthaquinones associated with the aflatoxin biosynthetic pathway, Bioorg Chem., 33, 426, 2005. 

Wogan. Structural identification of the major ENA adduct formed by aflatoxin Bj in vitro. Proc. Natl. Acad. Sci. U.S.A. 74 1870-1874, 1977. 

Peanut phytoalexins appear to be involved in resistance to drought-induced preharvest aflatoxin contamination of immature peanuts. Mature peanuts are considerably more resistant to environmentally-induced preharvest aflatoxin contamination of peanuts than are immature peanuts. The mechanism of this latter resistance is unknown. The identification of this resistance mechanism and other resistance may provide one approach to subsequent use of biotechnology to incorporate field resistance traits into commercially acceptable varieties. Biotechnology may also be a valuable approach to exploiting genetic resistance to preharvest aflatoxin found in wild species that have evolved in an arid environment. 

Fig. 4. Identification of GST subunits in rat liver cytosol. SDS-PAGE was performed in a 12% polyacrylamide resolving gel. Samples were run from the cathode (top) to the anode (bottom) and the gel was stained with Coomassie brilliant blue. The fractions are as follows (1) liver cytosol from normal rats, (2) liver cytosol from nodule bearing rats that had been fed aflatoxin Bj (2 ppm) for approx 16 weeks, (3) hepatic cytosol from normal rats that did not bind the glutathione-Sepharose affinity matrbt, (4) hepatic cytosol from aflatoxin Bj-treated rats that was not retained by the glutathione-Sepharose affinity gel, (5) affinity-purified GST from normal rat liver, and (6) affinity-purified GST from rat livers that contained aflatoxin-induced preneoplastic nodules. The positions of the Ya, Yb, Yc, and Yf subunits are shown. The horizontal arrow adjacent to track 1 shows the position where the Yf (or Yp) would migrate if present. These data are taken from Hayes et al. (H23).

Yu, J., Whitelaw, C. A., Nierman, W.C., Bhatnagar, D., and Cleveland, T.E., Aspergillus flavus expressed sequence tags for identification of genes with putative roles in aflatoxin contamination of crops, FEMS Microbiol. Lett., 237, 333-340, 2004. 

PRIETO, R., YOUSIBOVA, G.L., WOLOSHUK, C.P., Identification of aflatoxin biosynthesis genes by genetic complementation in an Aspergillus flavus mutant lacking the aflatoxin gene eluster, Appl. Environ. Microbiol, 1996, 62, 3567-3571. 

O BRIAN G.R., FAKHOURY, A.M., PAYNE, G.A., Identification of genes differentially expressed during aflatoxin biosynthesis in Aspergillus flavus and Aspergillus parasiticus. Fungal Genet. Biol., 2003, 39, 118-127. 

Harris, A.J., Shaddock, J.G., Manjanatha, M.G., et al. 1998. Identification of differentially expressed genes in aflatoxin Bl-treated cultured primary rat hepatocytes and Fischer 344 rats. Carcinogenesis 19(8) 1451-1458. 

Aflatoxins are naturally strongly fluorescent compounds, so the HPLC identification of these molecules is most often achieved by fluorescent detection. Reverse-phase eluents quench the fluorescence of AFBl and AFGl [60] for this reason, to enhance the response of these two analytes, chemical derivatization is commonly required, using pre- or postcolumn derivatization with suitable fluorophore, improving detectability. 

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