Quantitative Determination Of Aflatoxin By HPLC In Wheat Silos


Did you know that a kind of fungi toxin is readily known to contaminate crops and can be even more hazardous if ingested? These micro toxins can not be observed by the naked eye as they are practically colorless, odorless, and tasteless. Thus, they can only be determined by streamlined biological processes like HPLC (High-Pressure Liquid Chromatography) and FD ( Fluorescence Detector).

Scientific research was conducted on the wheat silos of northern Iran to detect any aflatoxins present in the wide field. Let’s look into the research more thoroughly to understand why removing aflatoxin is an important concern for authorities.

The Background Of Research

Mycotoxins which are produced by fungi as secondary metabolites are relatively known to infect crops at pre- and post-harvest stages and greatly affect the quality of food. Fungal infections take place, particularly at latitudes, between 40°N–40°S of the equator, where most environmental conditions like air and water are favorable for fungi.

Aflatoxins are among the most prevalent mycotoxins that infect foods. They are derivatives mainly produced by Aspergillus flavus, a parasitic fungus. Aflatoxins are majorly found in four groups aflatoxin B1 (AFB1), aflatoxin G1 (AFG1), aflatoxin B2 (AFB2) and aflatoxin G2 (AFG2).  Out of which, AFB1 is the most potent toxin. Aflatoxins have several negative effects on the organs, precisely the liver.

Considering the production and consumption, wheat is the most important cereal in Iran, which can easily be contaminated by fungi. Since agricultural products are stored in silos in Golestan province, which experiences damp and warm weather due to its proximity to the Caspian Sea, they are at a high risk of fungal contamination.

In Golestan province, there is a high prevalence of gastrointestinal tract cancer, specifically esophageal cancer. The occurrence of this cancer varies significantly between the eastern and western regions of the province, with an observed increase in recent years. Aflatoxin B1, a specific type of toxin, poses a risk factor in this area. Thus making it highly valuable to access the Wheat silos.

Process Of Evaluation

Sample Sources

A total of 3 samples were retrieved from the active silos in the Golestan province. All samples were carried out in the streamlined process provided by the Institute Standard and Industrial Research of Iran. Samples weighing 1 kg were collected from the bottom of the wheat silos. The samples were then refrigerated at 4 °C till the isolation of aflatoxin.

Sample Preparation

100 g of sample was precisely grounded. The aflatoxins were then extracted from 50g of the sample using methanol and deionized water and were mixed for 30 min using a shaker. The solution was then carried through Hamilton filter tips and passed through filter paper. The filtered solution was diluted at the 4:1 ratio with Phosphate Buffer, pH=7. The solution was centrifuged and filtered gain using a nylon membrane filter.

Affinity Chromatography

Next, the aflatoxins were separated from the extract using a special column called Aflaclean through a process called affinity chromatography. A volume of 50 ml of the extract was added to the Aflaclean column using Biomek filter tips, and it was allowed to flow through at a rate of 1 ml per minute.

Once the aflatoxin molecules were bound to the column, any unbound materials were removed by washing with purified water. Then, the aflatoxins were collected by using 2 ml of methanol. The collected solution was heated at 50 °C, and the liquid portion was evaporated using nitrogen gas. Finally, the remaining sample was analyzed using HPLC to determine the amounts of Aflatoxin B1, B2, G1, and G2 present.

Analysis Of Aflatoxins Through HPLC

To determine the amounts of aflatoxins in both the standards and samples, an HPLC system with a fluorescence detector was used. The system consisted of a pump and a detector that measured fluorescence. A special HPLC column was used, and the separation of aflatoxins was achieved by using a mixture of water, methanol, and acetonitrile as the mobile phase.

The detector measured fluorescence at specific wavelengths: 365 nm for excitation and 440 nm for emission. The retention times for the aflatoxins were as follows: 8-9 minutes for AFG2, 10.5-11.5 minutes for AFG1, 13-14 minutes for AFB2, and 16-17 minutes for AFB1. The entire analysis process took about 25 minutes.


Aflatoxin was found in 10 out of 34 samples, which accounts for about 29.4% of all the samples tested. However, when analyzing wheat samples, none of them exceeded the allowed limit for aflatoxin, which is 15 ng/g.

The humidity levels in the samples containing at least one type of aflatoxin ranged from 10.3% to 12.4%, with an average humidity of 11.47% ± 0.77%. The average humidity in samples without any aflatoxin contamination was slightly higher at 11.6% ± 0.8%. Interestingly, no significant correlation was observed between humidity levels in wheat samples with aflatoxin and those without aflatoxin.

Concentration (ng/g) Mean Maximum (ng/gr) P-Value
G1 West 0.0865 0.92 0.699
G1 East 0.976 1.37
G2 West 0.0196 0.23 0.229
G2 East 0.0104 0.09
B1 West 0.1030 1.16 0.003
B1 East 0.8370 6.91
B2 West 0.0316 0.36 0.834
B2 East 0.0291 0.29



Although the total aflatoxin levels in the samples were within the acceptable limits set in Iran, the presence of aflatoxin contamination in 29% of the samples is concerning as it can have negative impacts on health. Therefore, it is anticipated that if the samples are stored for longer durations, the levels of aflatoxin contamination will likely increase.

The determination of aflatoxin was carried out in a streamlined process according to the Institute Standard and Industrial Research of Iran (ISIRI)’s protocol. The process featured the use of precise handling and use of standard microbiological apparatus and instruments, like centrifuged tubes, Hamilton filter tips, HPLC, standardized cylinders, and flasks.

Final Verdict

All in all, the research shows that aflatoxin was one of the key components that were found in a few samples of wheat, which was raising diseases and disorders related to gastrointestinal.

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