Mycotoxin Risk Assessment: Telling the Full Story

The research is clear: producers of food and feed need to take the synergistic effects of mycotoxin co-occurrence into account.

Yet how do we get a sample to spill all its secrets? As a method of multi-mycotoxin analysis, LC/MS-MS is already showing great promise.

There are approximately 400 compounds of low molecular weight that are recognized as mycotoxins, each with its own toxic effects for humans and animals. Yet national and international regulations and recommendations typically cover only a handful of mycotoxins: aflatoxins B1, B2, G1, G2 and M1; fumonisins B1, B2 and B3; ochratoxin A, deoxynivalenol, zearalenone, HT-2 toxin and T-2 toxin. These mycotoxins are well characterized and frequently covered in available research literature.
What do we know about the other unregulated mycotoxins in a particular sample? What are the hidden risks of their co-occurrence? How do we get a sample to tell its full story? This article will investigate unregulated mycotoxins, discuss the effects of mycotoxin co-occurrence, and introduce a method for analyzing samples of grain and feed for the simultaneous presence of multiple mycotoxins.

Emerging and masked mycotoxins

Emerging mycotoxins are those that are potential candidates for regulation owing to their increasing frequency and their toxicological composition, i.e. their potential for harm to animals or humans that could consume them. Strategies to measure and confront them are in a nascent stage and are currently subject to rapid development. 
One particular subclass of unregulated mycotoxins is “masked mycotoxins.” Fungi release a wide range of metabolites, including the well known DON and ZON, into the plant as part of the infection process. Often, this host plant will then further modify the compounds coming from the fungus. This chemical modification provides the “mask” that obscures the true identity of the mycotoxin.
Masking, however, serves an important purpose for the host plant as it constitutes one of its major detoxification strategies. Usually, a glucose molecule or a sulfate is involved in the conjugation and detoxification. Although these masked toxins do not further harm the plant, their toxicity to humans and animals might reemerge when the added masking molecule is cleaved in the gastrointestinal tract of mammals during digestion (Figure 1). In plant breeding, the increasing occurrence and production of some masked mycotoxins could be linked to novel resistant breeds. Deoxynivalenol-3-glucoside, for example, has been reportedly linked to resistance against Fusarium head blight. Although Fusarium-resistant plants show lower levels of total DON, higher DON-3-Glu/DON ratios have been reported in such plants, showing an increased production of this masked mycotoxin.

Modified mycotoxins and their toxicity

The “modified mycotoxin” is a further term signifying the changes that mycotoxins can undergo. Modified mycotoxins refer to both the modification of a parent toxin molecule by the fungus itself and the masking of the toxin that occurs within the plant tissue. Another type of modification takes place in mammals when aflatoxin B1 is consumed through contaminated feed and converted to aflatoxin M1. This aflatoxin M1 migrates into the milk of lactating animals and is subsequently excreted with it. In addition, modifications of toxins can also occur during food processing, in particular heating and fermentation, increasing their prevalence. These modified mycotoxins might occur in relevant amounts in food and feed.
Of all mycotoxins, deoxynivalenol has been most researched with regard to frequently observed modifications. The modified forms of deoxynivalenol can be divided into two main groups: altered and masked forms. There are two main altered forms of deoxynivalenol secreted by the fungus itself: 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, as found in Fusarium-contaminated cereals. Plants are able to mask the deoxynivalenol to deoxynivalenol-3-glucoside and, as recent studies show, this may take on two sulfonated forms: deoxynivalenol-3-sulfate and deoxynivalenol-15-sulfate.
So what specific harm can come of modified, masked and other emerging mycotoxins? Modified mycotoxins can be either more or less toxic than their parent compounds. For example, they may be more bioavailable due to modifications. Toxicological data on modified mycotoxins are scarce, and current results and knowledge on the real risks and effects of these compounds are insufficient. This lack of knowledge makes it difficult to conduct a proper risk assessment. Nevertheless, there have been studies describing their potential threat to food safety. Masked mycotoxins can be “unmasked” in the digestive tract of animals and humans, releasing the parent compound with its toxicological effects once again. A similar situation exists with other emerging mycotoxins: toxicological data are scarce which makes it difficult to set up regulations and maximum tolerated limits to protect humans and animals from potential health risks.
As modified mycotoxins behave differently in their chemical reactions than their parent mycotoxins, they can be easily missed in routine analysis. Current detection methods for regulated mycotoxins in food and feed do not include routine screening for these modified mycotoxins as they are not covered by legislation. Such standard methods may show up as contamination levels below legislative limits, while contaminations from modified mycotoxins go undetected. This represents a correct result, but from a toxicological point of view the integration of modified toxins (e.g. as a sum parameter) would provide more sound data for risk assessment. Together, all these facts point to the possible hazards posed by modified mycotoxins to human health. Regulations on the maximum levels of modified mycotoxins as well as other emerging mycotoxins are currently under discussion at the European Food Safety Authority.

Mycotoxin co-occurrence

Now that we know that several mycotoxins are produced by the same fungi, it should come as no surprise that the converging effects of multiple mycotoxins have increasingly become the subject of research. Data collected in several independent studies in recent years show that agricultural commodities are often contaminated with more than one mycotoxin. The toxicological interactions of mycotoxins are typically synergistic: this means that the toxicological effect of two or more mycotoxin present in the same sample will be higher than the sum of toxicological effect of the individual mycotoxins. That said, the co-occurrence of mycotoxins can also have additive or, more rarely, antagonistic effects, by which their effects cancel each other out.

These synergistic effects vary from animal to animal and can be very complex. Figure 2 shows both the synergistic and additive effects of certain mycotoxins in poultry. Aflatoxin B1 (AFB1), for example, is a regulated mycotoxin that exhibits synergistic effects with diacetoxyscirpenol (DAS) and cyclopiazonic acid (CPA), both unregulated mycotoxins, and an additive effect with DON, another regulated mycotoxin. In swine, however, as shown in Figure 3, AFB1 has no known co-occurring effect with DON or DAS, instead demonstrating synergistic effects with T-2 toxin and ochratoxin.

These are just a couple of examples of co-occurrence. The increasing awareness for mycotoxin co-occurrence makes it necessary to develop multi-mycotoxin detection methods that simultaneously analyze several mycotoxins. In response to this need, Romer Labs has developed the Multi-Mycotoxin Analysis 50+ method.

This analysis gives a unique insight into the contamination pattern of a sample and quantifies more than 50 mycotoxins, including aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, zearalenone, and “masked” mycotoxins such as deoxynivalenol-3-glucoside.
A discussion of the advantages, limitations and development of the method with respect to the chemical diversity of analytes and the range of agricultural commodities that can be tested follows.

Multi-mycotoxin analysis

Analysts are increasingly turning toward LC-MS/MS (liquid chromatography/tandem mass spectrometry) as a chief method for detecting multiple mycotoxins. Analytical methods based on LC-MS/MS have become a powerful and state-of-the-art technique in the qualitative and quantitative analysis of mycotoxins over the last decade. This technique enables the simultaneous determination of a wide range of mycotoxins belonging to different chemical families within one single measurement: acidic (fumonisins), basic (ergot alkaloids), polar (moniliformin, nivalenol) and apolar compounds (zearalenone, beauvericin) can all be simultaneously quantified with LC-MS/MS. 
Further advantages of this method are its high sensitivity and selectivity, as well as the delivery of additional information about mass-to-charge ratios (m/z) and fragment ions of the analytes under investigation. 
The set-up of a multi-mycotoxin method based on LC-MS/MS usually follows a three-phase process on the way to implementation: method development, method optimization and method validation. These steps and parameters are summarized in Figure 4. During method development and optimization, the quality and reliability of the results should be evaluated carefully. For this purpose, analytical standards of the highest quality, with certified concentration and stated purity must be used. However, for certain analytes, analytical standards are not commercially available. In those cases, it might be possible to access standards that are still under research, or to work with available material that is less well characterized.

Method development

During the development of a method based on LC-MS/MS, the MS and LC parameters, as well as the sample preparation procedure must be optimized. To optimize the MS parameters, each target compound should be injected as a pure analytical standard directly into the mass spectrometer. This allows for the selection of the best ionization mode (positive or negative) and the most abundant precursor and product ions. 
Also, the chromatographic conditions can be adjusted in this phase: the ideal mobile phases and gradient as well as the optimal chromatographic column must be evaluated. 
Many multi-mycotoxin methods use a dilute-and-shoot approach: this refers to the practice of diluting the sample extract before injecting it into the LC-MS/MS. A clean-up step can also be implemented as part of the sample preparation. However, the mycotoxin pattern must not be altered during the sample clean-up, i.e. one must make sure that no mycotoxin of interest is retained by the material used for the clean-up. 

Method optimization

The optimization of the analytical method includes stability testing of the analytes in standard solutions and samples, as well as proofing selectivity and determination of the working range.

Method validation

Method validation is a prerequisite for the production of reliable results in terms of comparability and traceability. Method validation must be performed separately for each target analyte in all required matrices. Typical performance characteristics that should be evaluated during validation of a quantitative method are limits of detection (LOD), limits of quantification (LOQ), linearity, precision, selectivity, robustness, accuracy, matrix effects and recoveries. For Multi-Mycotoxin Analysis 50+, the validated matrices are wheat, maize, swine feed and silage.
Matrix effects are encountered when matrix components interfere with the ionization process of the target analytes. Matrix effects can have a considerable impact on the quantification of mycotoxins. Therefore, it is essential to determine and compensate for such matrix effects. This can be achieved by determining the apparent recovery followed by a mathematical correction of the results with this value, by matrix-matched calibration, or by applying isotope-labeled internal standards. The latter will lead to results with the highest degree of accuracy and reliability at minimal investment of time and cost per sample.
The method can be validated by spiking blank samples with each required analyte at a range of concentrations in replicate. When available, the trueness of the method should be confirmed using certified reference materials. Furthermore, matrix-matched 
materials and participation in proficiency testing enable additional quality assurance. 

Conclusion

The development of a multi-mycotoxin method based on LC-MS/MS shows great potential in addressing the risk of mycotoxin co-occurrence. As the 
research community acquires more and more know-ledge about the synergistic effects of multiple mycotoxins, including emerging and masked mycotoxins, validated analytical methods that can detect them within the same sample will only grow in importance. 
A vast number of different parameters that significantly influence the quality and reliability of the results of the LC-MS/MS method must be considered carefully for each analyte in each matrix separately. In addition, the chemical diversity of the mycotoxins necessitates making compromises during method development, which may be far from optimal for certain analytes. Moreover, the wide range of agricultural commodities as well as the varying concentration ranges and different occurrence distributions pose further challenges to method development and optimization. Nevertheless, the development of multi-mycotoxin methods fulfills an urgent need; advances in this technology will further extend its application.

This article was published in Spot On Special Issue: Mycotoxins

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  • BIOMIN World Mycotoxin Survey 2018


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