*3.7. Method Validation*

The method employed for the extraction and simultaneous analysis of AFB1, AFB2, AFG1, and AFG2 in the laboratory culture conditions was validated according to the AOAC Guidelines Appendix F [33], with slight modifications, by determining the recovery, precision, selectivity, linearity, and the sensitivity. A mixture of known concentrations of four AFs (500, 100, 50, 10, and 2.5 of each) were spiked into blank culture samples (submerged, slant, and solid state) for the recovery validation. Each concentration was spiked in triplicate, and the experiments were repeated three times within a day and repeated in 3 consecutive days by the same operator. Precision was demonstrated as repeatability, which was evaluated by calculating the relative standard deviation (% RSD) of the spiked toxins repeated within 1 day and over 3 consecutive days. Blank samples were prepared by inoculating non-aflatoxin-producing *A. flavus* NRRL 21,881 in submerged, solid, and slant cultures. The samples were then harvested, and AFs were extracted and analyzed by HPLC coupled with DAD and FLD. The selectivity of this method was assured as there were no interfering chromatographic peaks corresponding to the retention time of the four AFs. The linearity was demonstrated for the AFs in the range of 2.5 to 1000 ppb in three replicates. The calibration standard of each concentration was constructed using the peak-area ratio of the AFs versus the concentration of the analytes. The linear relationship was evaluated by the correlation coefficient, *y*-intercept, and slope of the regression line. The sensitivity of the method was determined by measuring the LOD and the LOQ on the basis of a signal-to-noise ratio (S/N) of 3:1 and 6:1, respectively.
