YAMAMOTO CO., LTD.

Mycotoxin

Aflatoxin B1, B2, G1, G2

Acute intoxication caused by aflatoxin affects the bile duct and hepatoma and can be fatal. Aflatoxin is also known to be the most carcinogenic of all natural substances. Taking even a minute amount of aflatoxin for a prolonged time can cause hepatic cancer.

Extraction, clean-up:
1) Extraction: Add 250ml of chloroform and 25ml of water to 50g of the sample, and perform shaking extraction for 30 minutes.
2) Filtering: Use a filter paper coated with a 1cm thick layer of diatomaceous earth to perform aspiration filtering.
3) Dehydration: Dehydrate the filtered solution with sodium sulfate anhydride.
4) Test solution: Fifty milli-litres of filtered and dehydrated solution are used for the test solution.

Silica gel chromatography:
1) Activation of silica gel: Activate the silica gel 60 for column chromatograph for one hour at 105℃, add 1% of water, plug it tightly and cool in a desiccator.
2) Preparation of the column: Fill a chromate-tube of 22mm inner diameter with i) 5g of sodium sulfate anhydride, ii) 10g of silica gel, and iii) 15g of sodium sulfate anhydride in this order, using chloroform.
3) Column loading and washing: Load the column with the test solution, and wash it with 150ml of n-hexane and 150ml of diethyl ether anhydride.
4) Elution: Dissolve it with 150ml of methanol-chloroform (3+97).
5) Evaporation to dryness: Dry the eluted solution under vacuum.
6) Test solution: Dissolve the concentrated-dried substance to 1ml of acetonitoril-benzene (2+98) for the test solution.

Thin layer chromatography (TLC):
Prepare testing equipment such as a developing tank, TLC plate, fluorescent testing lamp (365nm), and micro syringe.
1) Activation of TLC plate: Activate the Kieselgel 60 silica gel plate for one hour at 105℃ and keep it in the desiccator.
2) Preparation of developing solvents: Chloroform-acetone-n-hexane (85+15+20) in volume.
3) Spotting extracted solution: Spot both the test solution and the standard aflatoxin solution on the TLC plate.
4) Development: Perform saturated development using the developing solvent mentioned above.
5) Confirmation: After development, the plate is air dried, and the presence of aflatoxin is examined under a fluorescent testing lamp.
6) Presence of aflatoxin: If the presence of aflatoxin is confirmed, an additional quantitative measurement is performed by high-performance liquid chromatography.