Aflatoxin Assay for Ground Corn

Extraction of Aflatoxin

  • Grind dry kernels in a Romer mill and mix well
  • Weigh 20g ground kernels
  • Add 40ml 50% ethanol and shake for 2 minutes
  • Store at 4C until use

Dry sample and re-suspend

  • Add 200ul to 1.5ml tube and dry
  • Re-suspend in 20ul chloroform
  • Spot on TLC plate along with standards

Quantification of Aflatoxin using TLC

  • Spot standards of aflatoxin along with the sample to be quantified on the TLC plate
  • 10, 20, 50, 100, 500 and 1000 ng of aflatoxin can be used as a standard
  • Every plate should contain both the standard and the sample
  • After running the TLC, take a picture of it and use Imagej to quantify it


Using Imagej to quantify toxin

  • Use imagej to open the picture
  • Draw a rectangle over each spot on the TLC
  • On the tool bar, click on gel and open analyze
  • Using that, designate each lane as Lane 1,2,3 etc
  • After plotting the lanes, click on analyze
  • A peak will be obtained for each spot on the TLC, the base line of the peak can be adjusted to get the exact area
  • Go to the small wand on the toolbar (Next to the A) and click on it
  • It will give the area under each plot
  • On an excel sheet, plot the areas against known concentrations of aflatoxin to get a “Standard graph”
  • Use the standard graph to plot the area of the test (whose concentration is not known) and using the standard graph bring it down to the X-axis to find its concentration.