Determining aflatoxins in raw peanuts using immunoaffinity column as sample clean-up method followed by normal-phase HPLC-FLD analysis

This study was aimed at simplifying the conventional aflatoxin HPLC-FLD analysis procedure for determining aflatoxins B1, B2, G1, and G2 by utilizing the advantage of immunoaffinity column (IAC) and normal-phase chromatography. Normally, a derivatization procedure is needed to enhance the fluorescence signal of aflatoxin quenched by the reversed-phase solvent used as the HPLC mobile phase. However, the proposed mobile phase (toluene/ethyl acetate/methanol/formic acid, 90 mL/6 mL/2 mL/2 mL) considerably enhanced the fluorescence signal by eliminating the quenching effect. IAC is a fast and reproducible method for sample cleanup, but the miscibility of the eluate is low in the normal-phase solvent. The miscibility of the eluate in the normal-phase solvent was improved by replacing methanol with acetonitrile as the eluent, dehydrating water after IAC elution, and mixing with toluene before HPLC analysis. The results demonstrate that the timeconsuming procedures, such as nitrogen evaporation and iodine-derivatization, in the sample preparation were no longer needed, which improved the recovery and repeatability. The LOQ can be as low as 2.0-0.51.0-0.5 ng of aflatoxin B1-B2-G1-G2 per gram of peanut. All linearities (R2) of the four types of aflatoxin were greater than 0.999. The inter-day recovery rates of aflatoxin B1, B2, G1, and G2 at LOQ were 109%, 115%, 113%, and 77%, and the corresponding standard deviations were 12%, 8%, 5%, and 14%, respectively. The performance of the proposed method in analyzing raw peanuts was better than that of the official method (HPLCFLD combined with post-column iodine-derivatization) and can meet the US FDA's standard for examining human food. The shelf-life of the proposed mobile phase was prolonged to 2 weeks under room temperature, further saving the use of solvent. Each analysis needed less than 25 mL of organic solvent to complete. Furthermore, the method can determine AF concentration in raw peanuts contaminated with more than 4.00 ng/ g of total aflatoxins (containing 2.00 ng/g of aflatoxin B1, 0.50 ng/g of aflatoxin B2, 1.00 ng/g of aflatoxin G1, and 0.50 ng/g of aflatoxin G2).

Automated summary

This study aimed to simplify the conventional aflatoxin analysis method by utilizing the immunoaffinity column and normal-phase chromatography to detect aflatoxins B1, B2, G1, and G2. The optimized mobile phase enhanced fluorescence signal and improved sample cleanup method significantly increased recovery and repeatability. The proposed method outperformed the official method and met the US FDA's standards for examining human food, with a prolonged shelf-life of the mobile phase under room temperature.