| Summary: | High-performance liquid chromatography (HPLC) provides a quick and efficient tool for accurately characterizing aflatoxigenic and non-aflatoxigenic isolates of <i>Aspergillus flavus</i>. This method also provides a quantitative analysis of AFs in <i>Aspergillus flavus.</i> The method’s recovery was assessed by spiking a mixture of AF at different concentrations to the testing medium. The validity of the method was confirmed using aflatoxigenic and non-aflatoxigenic strains of <i>A. flavus</i>. The HPLC system, coupled with a fluorescence detector and post-column photochemical reactor, showed high sensitivity in detecting spiked AFs or AFs produced by <i>A. flavus</i> isolates. Recovery from medium spiked with 10, 20, 60, and 80 ppb of AFs was found to be 73–86% using this approach. For AFB<sub>1</sub> and AFB<sub>2</sub>, the limit of detection was 0.072 and 0.062 ppb, while the limit of quantification was 0.220 and 0.189 ppb, respectively. The AFB<sub>1</sub> concentrations ranged from 0.09 to 50.68 ppb, while the AFB<sub>2</sub> concentrations ranged between 0.33 and 9.23 ppb. The findings showed that six isolates produced more AFB<sub>1</sub> and AFB<sub>2</sub> than the acceptable limit of 5 ppb. The incidence of aflatoxigenic isolates of <i>A. flavus</i> in sweet corn and higher concentrations of AFB<sub>1</sub> and AFB<sub>2</sub> emphasize the need for field trials to explore their real potential for AF production in corn.
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