Validation and robustness testing of an OPLC method for the determination of aflatoxins in wheat

This paper describes validation of an OPLC procedure developed for the dete rmination of aflatoxins B-1, B-2, G(1), and G(2) in wheat. Samples were ext racted with 9:1 (v/v) acetonitrile-water and the extracts were filtered and evaporated to dryness. OPLC was performed with chloroform-toluene-tetrahyd rofuran, 15 + 15 + 1 (v/v), as mobile phase. Before the separation an OPLC prewashing step was necessary; this eliminated the time-consuming and costl y clean-up steps of other chromatographic methods. The validation procedure included tests on specificity and determination of the retention factor (R -F), resolution (R-S), asymmetry (A(S)), linearity, accuracy, precision, de tection limit (DL), and quantitation limit (QL) of the method. Depending on the anatoxin analyzed the results of the investigation were: 0.20 < R-F < 0.60, R-S > 1.7, 0.85 < A(S) <1.1, recovery > 84%, RSD < 10%. The DL of the method was 0.018, 0.100, 0.15, and 0.14 ng for anatoxins G(2), G(1), B-2, and B-1, respectively. A calibration curve was constructed for each anatoxi n by plotting spot area against amount of anatoxin applied to the plate. Th e robustness of the procedure was also determined and found be critically d ependent on the type of plate used (TLC or HPTLC).