Aflatoxins are natural mycotoxins produced by Aspergillus fungi. High temperatures and humidity favour the occurrence of moulds and therefore the production of aflatoxins. The contamination
of crops, nuts, dried fruits or vegetables, dried medicinal plants, and milk is quite common. Because of their strong carcinogenicity,
aflatoxins must be controlled in food and feeds.
Figure 1: Image of the derivatized plate under UV 366 nm.
This method is suitable for the quantification of aflatoxins B1, G1, B2, and G2 in tomato extract according to the Test for Aflatoxins (1) which limits aflatoxin B1 to 5 ppb and the sum of B1, G1, B2, and G2 to 20 ppb. Chromatography is performed on HPTLC plates according to Method II.
Required or Recommended CAMAG Devices
Automatic TLC Sampler 4 or Linomat 5, Automatic Developing Chamber ADC 2 or Twin Trough Chamber 20 cm × 10 cm, Visualizer,
TLC Scanner, and winCATS software.
Table 1: Track assignment.
Transfer 5 g of a representative powdered sample to a glass-stoppered flask. Add 20 mL of methanol and water (17:3). Shake
vigorously by mechanical means for 30 min and filter. Discard the first 5 mL of the filtrate and collect the next 4 mL portion.
Transfer the filtrate to a separatory funnel. Add 4 mL of sodium chloride solution (5 g of sodium chloride in 50 mL of water)
and 2.5 mL of hexane, and shake for 1 min. Allow the layers to separate and transfer the lower aqueous layer to a second separatory
funnel. Extract the aqueous layer in the separatory funnel twice, each time with 2.5 mL of methylene chloride, by shaking
for 1 min. Allow the layers to separate each time. Separate the lower organic layer and collect the combined organic layers
in a 50 mL conical flask. Evaporate the organic solvent on a water bath. Transfer the remaining extract to an appropriate
sample tube and evaporate to dryness on a water bath. Cool the residue.
If interferences exist in the residue, proceed as directed for Cleanup with immunoaffinity column (IAC); otherwise, dissolve the residue obtained in 200 μL of acetonitrile, and shake by mechanical means if necessary.
Figure 2: Densitogram of standards aflatoxin G2, G1, B2, and B1.
Cleanup with Immunoaffinity Column (IAC)
Dissolve the residue of the above sample solution in 5 mL of methanol and water (60:40) and then dilute with 5 mL of water.
Apply this extract onto a conditioned IAC. Rinse the IAC twice with 10 mL of phosphate-buffered saline (PBS) solution*, and
perform the elution slowly with 2 mL of methanol. Evaporate the eluate with nitrogen, and dissolve the residue in 200 μL of
Prior to conditioning, the IAC should be adjusted to room temperature. For conditioning, apply 10 mL of PBS solution on each
column and pass through at a rate of 2–3 mL/min by gravity. Leave 0.5 mL of PBS buffer on top of the column until the test
solution is applied.
Figure 3: Densitogram of a tomato extract sample (red) and the same sample spiked with 5 ppb of aflatoxins B1 and G1 (blue).
For this application note, the sample of tomato extract was extracted using an IAC from R-Biopharm.
Accurately weighed standard solutions containing 0.05 μg/mL aflatoxin B1 and aflatoxin G1 and 0.01 μg/mL aflatoxin B2 and aflatoxin G2 in a mixture of chloroform and acetonitrile (9.8:0.2) were prepared.
Figure 4: Calibration function for aflatoxin B1 measured at 366 nm. Regression via area y = -113.971+1.931x+-0.001x2; r = 0.99998; sdv = 0.84%.