Method Validation for the Quantitative Analysis of Aflatoxins (B1, B2, G1, and G2) in Rice by HPLC with Fluorescence Detection

Aflatoxins are a group of toxic metabolites produced by species of Aspergillus, which were found worldwide in air and soil. Aflatoxins commonly found are aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2). AFB1 is the most potent of all aflatoxins known to date and is generally found in the highest concentration in food and animal feeds. Aflatoxins occur naturally in food substances, and can colonize these substances either during the growth period or in storage. Worldwide, rice is one of the most important staple food for more than half of the world’s population. Usually, rice cultivation is conducted in subtropical/tropical environments, which are characteristically warm and humid. Furthermore, rice is generally dried after harvesting and under inappropriate storage conditions, rice can be an ideal substrate for mycotoxin-producing fungi [1].After a deep research, the author has concluded that few studies about the Iranian rice have provided a fully validated method of analysis. Analytical methods used by enforcement laboratories in order to implementation of legislation, must be subject to validate procedures. In order to ensure the results of the testing method, these methods need to provide accurate and reproducible results both within and between laboratories. This is especially important in view of legal actions and trade specifications, as well as monitoring and risk-assessment studies. This study aimed to validate a methodology for the determination of aflatoxins B1, B2, G1 and G2 in rice, using A KOBRA® Cell derivatization system and quantification by HPLC with fluorescence detection [2].The extraction of AFs from rice were performed by this method: blending 50g of sample rice with 200ml of methanol:water (80:20, v:v) in 5 min.Then the extract was filtered and 20 ml of that were transferred to a flask and diluted to 130 mL with PBS buffer (pH 7.4 ± 0.1). The diluted extract was passed through an AflaTest immunoaffinity column at a flow rate of 2-3 ml/min. Subsequently, 1.5 mL of methanol were applied to the column and finally samples were eluted by gravity into a glass vial. 1.5 milliliter of water was added, 50 μL were injected into the HPLC system.The modified methods were validated by measuring the specificity, selectivity, linearity, sensitivity, accuracy, repeatability, reproducibility, recovery, LOD, and LOQ parameters.High linearity was obtained over a dynamic range of 0.4–7.2 μg/kg for each aflatoxin. Recoveries varied from 92.21 to 108.28 %, with reproducibility standard deviations lower than 8.63% and repeatability standard deviations lower than 5.16 % Limits of quantification (LOQ) were 0.4 μg/kg for B1, B2, G1 and G2. Limits of detection (LOD) were 0.13 μg/kg.