Analysis of Mycotoxins in
feed materials

Mycotoxins are toxic substances produced by mycotoxigenic fungi in agricultural products, mainly in cereals.

From the food safety point of view, the most important mycotoxins are:
  • Aflatoxins
  • Ochratoxin A
  • Zearalenone
  • Fumonisins
  • Trichothecenes
Mycotoxins are emerging as a major contaminant of feed and food.
Josefa Tolosa
Degree in Veterinary Medicine and Doctor in Food Science
Associate Professor. University of Valencia.
Food Chain

Cereals and Mycotoxins

Cereals are the main raw material of animal feed (Santos et al., 2011), that is why mycotoxins have become one of the greatest challenges of the livestock industry, affecting at the same time other parts of the food chain, such as stores, manufacturers and farmers.

Figure 1. Exposure to mycotoxins through ingestion in humans and animals (Tolosa, 2017)
Food Chain
Nowadays, one of the problems of great interest is the presence of mycotoxins in animal feed, as these can be transferred to tissue and derived products (meat, milk, eggs) of animals fed with contaminated feed (Figure 1).
Food security is a great challenge for the livestock sector because it affects animal health and the food chain
Analysis

Rapid methods of detection

The obligation to comply established regulatory limits has promoted the development of analytical techniques for the analysis of mycotoxins in food and feed, in compliance with control systems and established quality requirements.
Among the rapid methods are those qualitative or semiquantitative easy to handle methods allowing quick and economic analysis with low instrumental component, primarily used for screening of a large number of samples (González-Sapienza & Venancio, 2011).

Most of the rapid methods are immunochemical assays based on the use of antibodies as specific recognition elements of the target mycotoxin, commonly used as routine analysis in feed mills.

Immunochemical methods

Operational principle of these methods is the antigen-antibody reaction, where the antigen is a modified mycotoxin, which, once produced, becomes a signal allowing quantitation thereof.
The main problem with immunochemical methods is that positives must be confirmed by techniques with better selectivity, such as mass spectrometry (MS) to be legal.
Immunoassays

The most commonly used technique is the Direct Competitive Enzyme Linked Immunosorbent Assay (ELISA) (Figure 2), marketed as kits composed of microplates covered with the mycotoxin antibody, reagents and standards necessary to perform the analysis.

Advantage

They provide important simplification of the preparation stage of the samples, since they require smaller sample volumes and simpler purification procedures.

Quick, simple, specific and quantitative method.

Disadvantages

Cross reactions between mycotoxins of the same group or interferences with the matrix.

Figure 2. Principle of competitive ELISA for mycotoxin analysis (Ortiz et al., 2014).
ELISA

This technique is based on the ability of a specific antibody to distinguish the three-dimensional structure of a given mycotoxin and it requires only a simple extraction of the sample with a solvent.
After the development of the kit, a colorimetric reaction inversely proportional to the concentration of mycotoxin in the sample is generated, which is measured by the ELISA reader at an absorbance of 450 nm.

Membrane-Based Immunoassays: Flow-Through / Lateral Flow Test

This is a direct competitive ELISA consisting of strips composed of one block where the sample is placed, of another block where the conjugate is placed, of a membrane, of an adsorbent block and of an adhesive base, wherein the anti-mycotoxin antibody binds to the surface of a membrane (Figure 3).

It is a semiquantitative method, very simple, fast and stable, which makes it very useful technique for decision making in the field and in the factory.

Figure 3. Flow-Through Test
Flow-Through Test
Extraction Using Immunoaffinity Columns

Immunoaffinity columns have been commonly used due to their high selectivity and the fact that they are easy to use. The procedure consists of preconditioning the columns, which is followed by the addition of the extract to be analysed. Mycotoxin specifically binds to the monoclonal antibodies present in the column, while the rest of the compounds are eliminated after the addition of a washing liquid.

After the washing, mycotoxins are eluted by the addition of a solvent that causes denaturation of antibodies (Figure 4) (Soriano et al., 2007). Advantages of this method are its high specificity, it does not require very clean initial samples and it provides high recoveries and purity of the extracts. However, they are not reusable and they are relatively expensive.
Figure 4. Immunoaffinity columns application scheme
Immunoaffinity
Although the method of immunoaffinity columns was originally developed as a fluorometric quantifying technique, the columns are currently used for purification and concentration of mycotoxins for their subsequent detection by liquid and / or gaseous chromatography.

Instrumental methods
Chromatographic techniques



High-performance liquid chromatography
High-performance liquid chromatography (HPLC) represents the technique of choice for the analysis of mycotoxins or for confirmation of positive results by ELISA:
  • It is sensitive
  • Reproducible
  • Accurate
  • Has a higher degree of automation
The most widely used absorption and emission spectrometric techniques are ultraviolet (UV) and fluorescence (FL) detection, the latter being preferred when mycotoxins exhibit natural fluorescence.
However, the coupling of HPLC to MS detectors has allowed the development of new methodologies for the detection and quantification of mycotoxins and the establishment of multiresidue methods covering mycotoxins of different families, which allowed it to become one of the methods of choice for carrying out multi-mycotoxin analysis (Tolosa, 2017).
All of this, combined with low quantification limits and, in particular, the fact that it unequivocally allows confirmation of the presence of mycotoxins is increasingly making this technique considered as the best option for mycotoxin analysis.
Gas chromatography (GC)
Gas chromatography (GC) is used less frequently because most of the mycotoxins are not sufficiently volatile and, therefore, have to be derivatized, which increases the time of analysis.
The main group of mycotoxins analysed by GC are trichothecenes, obtaining detection limits and suitable variation coefficients prior silanisation reaction (Rodriguez-Carrasco et al, 2012;. Escrivá et al, 2016.).
The use of chromatographic techniques requires prior preparation of a sample using different methods of extraction and purification, depending on the mycotoxins to be analysed and the food matrix, such as immunoaffinity columns or other procedures based on solid-liquid (Quechers, DMFS, etc.) or liquid-liquid (DLLME) extraction.
The cost of the equipment and its maintenance is high, and it requires high degree of preparation of the analyst, which is why these techniques are not used as a first option at the field or the factory level when fast results are required, but rather as a confirmation and/or quantification technique.



Micotoxicosis prevention
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