Aflatoxins, toxic secondary metabolites produced by fungi of the Aspergillus species, are regulated in over 100 developed countries, and nearly 200 AOAC Official Methods for their detection are published. Validation of such methods is important to ensure the safety of the food supply, and key areas related to maximizing the recovery of aflatoxins remain under-investigated. Accordingly, variables associated with extraction and purification of aflatoxin extracts from peanut meal, the solids generated from peanuts crushed for oil, prior to high-performance liquid chromatography analysis were tested. Sample:solvent ratios were evaluated in the range of 1:2 to 1:8 using 60% aqueous acetonitrile or 80% aqueous methanol.  For this highly contaminated material, saturation of curves was achieved at 1:4 and 1:6 ratios for methanol and acetonitrile, respectively. Acetonitrile extracted 2.3 times the amount of aflatoxins as methanol; however, when peanut meal was pre-soaked at alkaline pH (10.8), protein was hydrolyzed and methanol extracted as efficiently as acetonitrile suggesting that protein denaturation facilitates aflatoxin release. Furthermore, two common purification columns, AflaTest� (immunoaffinity) and Florisil� (silica-gel) were compared using aqueous acetonitrile extracts (1:4 sample:solvent ratio). AflaTest� and Florisil� columns recovered equivalent amounts of aflatoxin; however, initial analysis of the flow-through from the Florisil� column revealed a significant amount of aflatoxins.  Further analyses of the flow through composition, including HPLC-MS, is underway to confirm this finding. A key step in AflaTest� purification is dilution of the extract prior to column loading. A dilution of at least 2.5x with water was required to maximize recovery.  At this dilution, extract load volume did not affect recovery.  Furthermore, addition of 2% acetic acid to aflatoxin standards was required to prevent degradation while running standard curves.  These studies highlight the importance of optimizing extraction and purification procedures for analysis of aflatoxins, especially in highly contaminated materials, for consistent and accurate quantification.

Improvement of extraction and purification of aflatoxin from highly contaminated peanut meal prior to HPLC quantification

247th National Meeting (2014)

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