Mycotoxins analysis
Essential pillar for mycotoxins
control and prevention

Analysis of raw materials for mycotoxin contamination is vital. Therefore, Patent Co. uses a fast and simple multi-mycotoxin UHPLC method for the accurate determination and quantification of all regulated mycotoxins in feeds by liquid chromatography coupled with tandem mass spectrometry.

Jog Raj, Hunor Farkaš and Marko Vasiljević

PATENT CO, Vlade Cetkovica 1A 24 211 Misicevo., Republic of Serbia

Adapted from: The importance of mycotoxins analysis. All About Feed

Analysing incoming feed materials for mycotoxin contamination is crucially important as feed can be affected by climate, agricultural practices and local legislation on mycotoxins.

In a 2017 survey, corn harvested in Serbia and Bosnia and Herzegovina was contaminated with high levels of Aflatoxin B1, Fumonisin B1, and Fumonisin B2.

The global use of feed materials in the production of animal feed is increasing the risk of chemical and microbiological contaminants in food-producing animals.

The feed can be contaminated with microorganisms, mycotoxins, animal by-products, organic pollutants and toxic metals.

This contamination of animal feeds has a negative effect on both animal and human health. Among these, mycotoxins are emerging as a major contaminant of feed and food.

Variations in mycotoxins are increasing the importance of analysing raw materials before they enter the feed chain. Photo: Mirko Nahmijas

Mycotoxins are produced as secondary metabolites by various fungi. The major mycotoxins producing fungi are Aspergillus, Fusarium, and Penicillium.

Aflatoxins, ochratoxin A, fumonisins, deoxynivalenol, T-2 toxin, and zearalenone are the most common mycotoxins found in food and feed samples.

Many food and feed samples can become contaminated with mycotoxins before harvest, during transport and during their storage.

Commodities and products frequently contaminated with mycotoxins and used in animal feed include corn, wheat, barley, rice, oats, nuts, milk, cheese, peanuts and cottonseed, etc.

Mycotoxins produce a wide range of adverse and toxic effects in animals affecting their overall health and productivity.

Mycotoxins cause mycotoxicosis and cause significant economic losses in animals due to reduced productivity, increased disease incidence and decreased reproductive performance.

The mycotoxins of most concern due to their toxicity and occurrence are aflatoxin (AFB1), deoxynivalenol (DON), ochratoxin A (OTA), zearalenone (ZEN), fumonisins (FB1 and FB2) and T-2 toxins.

Regulations for major mycotoxins in the food and feed commodities exist in at least 100 countries. Most of these regulations are related to aflatoxins and the maximum tolerated levels differ greatly among countries.

These variations in tolerated levels of mycotoxins and non-regulation of other mycotoxins in other countries are posing a big challenge for the animal feed industry and these variations are increasing the importance of mycotoxin analysis of incoming raw materials before they enter the food/feed chain.


To determine whether feed material and other commodities are contaminated with mycotoxins, they must be tested for mycotoxins.

Proper sampling procedures are a pre-requisite for obtaining reliable results because of the heterogeneous distribution of mycotoxins in grains and other commodities.

There are many methods available for the detection of mycotoxins. Conventional methods for mycotoxins include ELISA, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and gas chromatography (GC).

Most of these methods employ a solid phase column clean-up of extracts and immunoaffinity techniques to remove interferences to improve the measurement of mycotoxins.

⇰ ELISA is a method of choice where rapid analysis is required but requires confirmatory analysis by LC-MS/MS.

⇰ LC-MS/MS is the most sensitive and preferred method of analysis for mycotoxins in food and feed samples.


Since it is necessary to test animal feeds for mycotoxin contamination, Patent Co is using a fast and simple UHPLC-based multi-mycotoxin method for the determination and accurate quantitation of all mycotoxins (Aflatoxin B1, B2, G1, G2, Deoxynivalenol, Zearalenone, Fumonisin B1 and B2, T-2, HT-2, and Ochratoxin A) regulated in feed, by liquid chromatography coupled with tandem mass spectrometry.

The method is based on a ‘dilute and shoot’ principle.

It involves two step extraction and centrifugation of the extracts. To compensate the matrix effects in electrospray ionization, the extracts are mixed with [13C] labelled internal standards for each group of mycotoxins (13C AFB1, 13C DON, 13C ZEN, 13C OTA, 13C FB1, and 13C T-2) before injection into LCMS/MS.

The method was successfully validated on corn, compound feed, wheat, barley, soya meal, wheat bran, sunflower meal and TMR.

Method performance parameters were obtained by in-house validation.

The blank samples were spiked with a mixture of 11 mycotoxin standards on two levels (LOQ and 10xLOQ) in 12 replicates. The RSDr of the method were between 2.5% and 13.4% and the apparent recoveries were between 62% and 115% for all analytes.
It was therefore concluded that the ‘dilute and shoot’ method with the addition of [13C] labelled internal standard is capable of determining all EU-regulated mycotoxins in animal feed and compound feed.


A comparison has been made of the data obtained on a global level in 2018 with the results obtained in 2019 (Figure 1).

  • In 2018, 95% of the samples were contaminated with one or more mycotoxins.
  • In 2019, 92 % of the corn samples were contaminated with one or more mycotoxins.

Figure 1. Comparison of the number (%) of mycotoxins in global corn samples in 2018 and 2019

The data in Figure 2 shows that the mean FB1, FB2, DON, ZEN, were higher in 2019 when compared with those present in the corn samples in 2018.

Figure 2 data also shows that the mean contaminations of AFB1, OTA, T-2 and HT-2 were lower in the 2019 samples when compared with the 2018 survey. There was thus a reduction in AFB1, OTA and type A trichothecenes in the 2019 survey.

Figure 2. Comparison of the mean contamination levels (ppb) for each mycotoxin in the corn samples examined in 2018 and 2019.

The data in Figure 3 shows the higher occurrence of FB1 and of HT-2 toxins in the corn samples in 2019 when compared to those in 2018.

This contrasts with the predominant occurrence of the mycotoxins AFB1, OTA, ZEN, DON and T-2 toxins in the global corn samples in 2018v2019 samples received.

Figure 3. Percentage of toxins detected in corn samples in 2018 and 2019.

Figure 4 shows a comparison between the maximum of positive samples for different mycotoxins in 2018 and 2019.

Figure 4. Comparison of the maximum of positives detected in corn in 2018 and 2019.


When comparing the survey results in 2018 with the 2019 corn survey the following differences were found:

  • OTA was detected in corn samples in 2018, but not in 2019

  • There was a higher occurrence of Fumonisins, and with higher contamination levels in 2019

  • In the 2018 survey, there were more Aspergillus-related mycotoxins when compared to 2019

Read more about mycotoxin contamination in corn:

Multiple mycotoxins detected in corn harvested in 2019

Micotoxicosis prevention
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