Assessment of aflatoxins in pigmented rice using a validated immunoaffinity column method with fluorescence HPLC

The determination of aflatoxins B1, B2, G1 and G2 in a variety of rice types was carried out using an immunoaffinity column (IAC) clean-up procedure followed by liquid chromatography (LC) with fluorescence detection. The sample was blended at high speed to form a fine powder and a test portion was extracted with methanol–water (60 + 40) using a high-speed blender. The filtered sample extract was diluted with 15% Tween 20 in phosphate buffered saline solution, and applied to an immunoaffinity column. Tween 20 is an essential part of the method to improve recoveries which were otherwise adversely affected by pigments in the rice. Aflatoxins were removed with neat methanol, and then directly analysed by reversed-phase LC with fluorescence detection using post-column bromination (Kobra cell). Test portions of black and polished rice were spiked with a mixture of aflatoxins, giving recoveries for individual and total aflatoxins ranging from 75.2 to 94.7%. Triplicate inter-day analysis at two levels gave relative standard deviations for repeatability (RSDr) averaging 1.6% for total aflatoxins and 1.8% for aflatoxin B1. A small survey of rice obtained from local markets in China found only one sample of red kojic rice which contained 2.9 μg/kg of aflatoxin B1.