DLLME procedure
For DLLME, 5 mL of phosphate buffer saline (pH 7.4) containing 500 µL acetonitrile (disperser solvent), 500 µL NaNO3 10% (w/v) and 2.5 ng AFs stock, was placed in a 10 mL screw cap glass test tube with a conical bottom. Then 170 µL of chloroform (extracting solvent) was rapidly injected into the sample solution using a 500 µL syringe resulting in a cloudy solution. The aflatoxins were extracted into the fine droplets of CHCl3. The solution was then centrifuged at 5000 rpm for 3 min, and the dispersed fine droplets of CHCl3 were deposited at the bottom of the conical test tube. After removing the aqueous phase, the sediment phase was transferred into a boiling water bath for evaporation of chloroform and then, dissolved in 100…show more content… The mixture was vortexes so that the sample particles were completely dispersed in the extract solvent. After shaking at 400 rpm, it was centrifuged at 5000 rpm for 3 min and then filtered through a pre-folded filter paper. 20 mL of the obtained filtrate was pipetted and placed in a 125 mL Erlenmeyer flask, diluted with 130 mL water and filtered again through a glass microfiber paper. Immunoaffinity column (IAC) was equilibrated at room temperature for at least 15 min before use. The top cap of the column was removed and the column was connected to a reservoir of column manifold. Seventy mL of the filtrate was collected into a 100 mL graduate cylinder (the final weight after extraction stages was 2.33 g) and immediately submitted to the IAC. Then the bottom cap of the column was removed and the liquid in the column was let flow through until it reached a height of 2 mm. Seventy mL of the filtrate was now added to the column reservoir and let flow under gravity. Similarly, 10 mL washing solution was also added to the column reservoir. Using a 10 mL syringe to pass air through the column, the remaining few drops of solution were collected. Now a 10 mL screw cap glass test tube was placed under the column and elution was carried out using 5 mL of an aqueous solution (pH 7.4) containing 500 µL…show more content… The extraction solvent should have special characteristics such as higher density than water, high extraction capability of the interest compounds and low solubility in water. Four solvents, i.e., carbon tetrachloride, chloroform, dichloromethane and chlorobenzene were tested for the extraction of the aflatoxins. The results demonstrate that chloroform provided higher extraction efficiency than other organic solvents. This may be attributed to the greater polarity of chloroform relative to other solvents, leading to a higher solubility towards the aflatoxins and therefore higher extraction