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Scott Pritchett

Modern Mycotoxin Testing: How Advanced Detection Solutions Help Protect Brands and Consumers

By Scott Pritchett
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Scott Pritchett

Mycotoxins are toxic compounds produced by several types of fungi. These mycotoxin-producing fungi grow on a variety of crops and foodstuffs, such as cereals, nuts, and coffee beans, contaminating up to 25% of the world’s crops every year.

In humans, ingesting even small amounts of some mycotoxins can lead to acute poisoning—research has also linked mycotoxin ingestion to long-term effects such as cancer and immune deficiency. In livestock, the situation is similar with mycotoxin exposure responsible for a greater incidence of disease, poor reproductive performance, and suboptimal milk production. With the health consequences so severe, it’s easy to see why mycotoxin contamination can harm the brand reputation of food producers and suppliers.

Mycotoxin contamination also poses a significant economic risk. The U.S. FDA estimates that mycotoxin contamination is responsible for an estimated annual crop loss of $932 million while, the Food and Agriculture Association estimates toxigenic fungi drive annual food and food product losses of ~1 billion metric tons, which includes losses due to reduced livestock productivity.

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To minimize the risks posed by mycotoxins, robust mycotoxin testing is essential. Through rigorous testing, food suppliers can identify and remove unsafe products in the supply chain and indicate where preventive measures may need strengthening. Global regulations permit very low maximum levels of mycotoxins in foods, and international trade regulations make testing food and animal feed for mycotoxins a critical step before export.

Mycotoxins: A Significant and Growing Analytical Challenge

Mycotoxin testing is complex. Laboratories tasked with the endeavor face several hurdles, including:

  • Varied and complex matrices. Analyzing food samples for low levels of contaminants is inherently difficult, as complex matrices contain a myriad of other compounds that can interfere with analyte detection.
  • Greater testing burden than other contaminants. For example, with pesticides, testing after crop harvest is sufficient to ensure food safety, as foodstuffs are unlikely to acquire pesticide contaminants beyond this point. However, foods can acquire mycotoxin contaminants at several points after crop harvest—for instance, during transportation and storage. Testing at multiple points in the supply chain is therefore needed, which demands testing be easy to deploy, efficient, and cost-effective.
  • New, emerging threats. Mycotoxin contamination concerns go beyond the ‘big six’ classes most commonly encountered (aflatoxins, ochratoxin A, patulin, fumonisins, zearalenone, and nivalenol/deoxynivalenol). Emerging mycotoxins—lesser-known, novel mycotoxins neither routinely determined nor regulated—pose a threat to human and animal health, too. But new and emerging mycotoxins are not often reported or monitored due to limitations of current ELISA-based testing technology.
  • A warming climate. As average global temperatures rise, more regions will offer the warm, humid environments in which mycotoxin-producing fungi thrive. Some testing labs are already detecting classes of mycotoxins previously limited to other geographies. Moreover, rising temperatures may create ideal conditions for new mycotoxin-producing fungal strains and different combinations of mycotoxins. Thus, labs will increasingly need the flexibility to accommodate an expanding menu of known and unknown mycotoxin contaminants and mycotoxin combinations.
  • Broader, tighter regulations. Regulatory bodies are continually reducing the maximum permissible mycotoxin levels in food. For instance, in August 2022, the European Commission lowered the maximum levels of ochratoxin A in certain foodstuffs. Regulatory bodies will likely also expand analyte panels to accommodate new, emerging threats. Unless a laboratory’s testing equipment has sufficient sensitivity and flexibility to meet these continually changing requirements, labs may face unnecessary additional expenditure on new technologies each time regulations change.

Accordingly, to meet the needs of today while best positioning themselves for tomorrow, mycotoxin testing labs need testing methods that are highly sensitive, with the flexibility to quantify multiple known and unknown analytes in complex matrices. To meet ever-growing demand for efficiency, these methods should also be easy to use, and maximize lab productivity.

The Promise of Advanced LC-MS Solutions

Thankfully, advanced, high-throughput liquid chromatography mass spectrometry (LC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) solutions can alleviate these challenges.

For example, Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERs) sample preparation kits can accelerate and simplify sample preparation across a variety of matrices prior to LC-MS analysis, helping facilitate high-throughput multi-residue analysis. To improve analysis of low abundance analytes in complex matrices, automated solutions are available that perform analyte pre-concentration and sample clean-up online, offering analytical confidence and greater speed compared to offline methods. Similarly, robust, high-performance liquid chromatography (HPLC) and ultra-HPLC (UHPLC) solutions can better resolve analytes in complex matrices, maximizing instrument utilization, and unlocking superior laboratory throughput.

When it comes to mass spectrometry (MS) systems, more productive, confident targeted compound quantitation is possible with advanced triple quadrupole MS (QQQ) instruments. QQQ systems offer analysts high sensitivity, selectivity, and specificity, making them ideal for the detection of multiple low-level compounds in the most challenging matrices. In addition, the fast data acquisition speeds and rapid polarity switching of these systems mean labs can greatly improve their workflow productivity. The latest QQQ systems are also easy to use, require minimal training, and facilitate streamlined method creation and optimization, enabling labs to better keep pace with changing regulations and emerging threats.

The advent of orbitrap mass spectrometry has transformed analytical testing workflows across a range of applications, including mycotoxin testing. Orbitrap mass spectrometers offer ultra-high resolution (figure 1) and accurate mass measurements, together with high dynamic range. These instruments can, therefore, better resolve the lowest-level analytes from background interferences in crowded matrices, as well as elucidate the fine isotopic structures needed for more confident analyte identification. Orbitrap instruments have significant productivity benefits too, being able to perform both quantitative and qualitative analyses of multiple analytes in a single platform, and often in a single run—all while maintaining high sensitivity.

Orbitrap curve
Figure 1: Across a broad m/z range, orbitrap mass spectrometers offer superior resolution relative to other mass spectrometry systems, such as quadrupole time-of-flight (Q-TOF) instruments.

Perhaps most important, though, is the value of orbitrap systems for unknown analysis. Orbitrap systems can generate full-scan high-resolution accurate mass data during untargeted analysis, enabling analysts to capture information from all ions in the run. When this data is compared against extensive high-resolution spectral fragmentation libraries (such as the mzCloud), labs can more easily and confidently identify novel compounds such as emerging mycotoxins. Even when no direct spectral match is available, analysts can now tap into advanced data analysis algorithms that provide the best candidate structures for unknown compounds.

Toward Multi-residue, Multi-panel Workflows

Recent studies have demonstrated the success of several advanced LC-MS and LC-MS/MS solutions and workflows for fast, economical, and highly sensitive multi-residue mycotoxin analysis. For example, one recent study looked at quantifying 48 myco- and phytotoxins (either currently regulated or under discussion for regulation in the EU) in cereal in a single analytical run. In the experiment, researchers used a UHPLC system coupled to a QQQ instrument, and cereals were extracted with acetonitrile/water, followed by evaporation and sample reconstitution.

The results demonstrated that sample preparation is simple, fast, and economical for this method. And, for all legislated mycotoxins, the limits of quantitation were lower than the maximum residue limits (MRL) established by regulations. Researchers also noted good precision and reproducibility across five replicates at the limit of detection. The authors therefore concluded that this method is suitable for the quantitation of all 11 legislated mycotoxins and 37 more that are either already legislated in feed or are prospects for further legislation.

Another study demonstrated the value of an orbitrap-based workflow for efficiently detecting a range of unknown food contaminants, including mycotoxins. In the study, researchers analyzed vegetables, fruits, nuts, and cereals, preparing samples using the Swedish ethyl acetate method (SweEt). In terms of equipment, the team used an UHPLC system connected to an orbitrap mass spectrometer, and analyzed data using a qualitative software tool. To help identify unknown compounds, the software used isotopic pattern recognition, fragments, and isotope distribution data to search spectral libraries for matches.

The results showed SweEt to be an effective generic sample preparation approach to analyze different compound classes, and the workflow enabled the team to successfully identify many unwanted contaminants, including pesticides, mycotoxins, and food additives. As a result, the researchers concluded that the method was a suitable and efficient way to find unexpected and unwanted multi-group analytes in complex food matrices. Researchers have increasingly sought such multi-group analysis methods in recent years, owing to the significant efficiency gains they can offer to analytical labs.

Meeting the Needs of Modern Mycotoxin Testing

Mycotoxin contamination in food has significant consequences for human and animal health, as well as the reputation of food producers and suppliers. But mycotoxin testing is inherently complex, and several factors make testing increasingly difficult—from emerging mycotoxin threats to tightening regulations and growing pressure for greater efficiency.

Advanced LC-MS and LC-MS/MS solutions have the potential to address these challenges and transform mycotoxin testing workflows, delivering unprecedented sensitivity, confidence, and productivity. By adopting these high throughput, high-resolution solutions, testing labs can better meet the needs of today, while optimally positioning themselves for the future. Ultimately, these advanced approaches will help ensure the safety of the global food supply, for a healthier, safer world.

Rapid and Robust Technologies Improve Sample Preparation for Analyzing Mycotoxins

By Olga I. Shimelis
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Mycotoxins are produced as secondary metabolites by various mold species during the growth and harvest of grains, fruits, nuts and condiments. Their production is directly related to the dry/wet weather conditions during the growing season. Mycotoxins are very stable compounds and are not easily removed during storage, processing and preparation of raw agricultural commodities.

Mycotoxins & Grains
Mycotoxins can be found in a variety of grains.

Different classes of mycotoxins are distinguished on the basis of the structural similarity and originating mold species. For example, more than a dozen different aflatoxin compounds exist but only five of them are routinely tested (aflatoxins B1, B2, G1, G2, and M1). Aflatoxin B1 is of particular interest because it is listed as a Group 1 Carcinogen by the International Agency for Research on Cancer (IARC). Aflatoxin M1 is a metabolic product that can be present in milk upon ingestion of aflatoxin B1 by an animal. Aflatoxins are ubiquitous in important agricultural commodities including maize and peanuts, and are among the most studied mycotoxins.

Deoxynivalenol (DON) is produced by a different fungi species. It is prevalent in cereal crops grown under wet conditions and temperatures above 15o C (60o F). Chronic exposure of livestock to DON may result in slowed growth, impaired immune function and reduced rates of reproduction, particularly in non-ruminants.

Mycotoxins were discovered as the cause of poisoning outbreaks in both humans and farm animals in the mid-20th century. Since then, multiple government regulations were established to control the presence of these toxic compounds in food and feeds. For example, harvested grains are checked for mycotoxin contamination using rapid field screening methods prior to grain deposition into silos. If contamination is found, the crops are sent to an analytical laboratory to perform the confirmation analysis. Liquid chromatographic methods were often used for such analysis with both fluorescence and UV detection. In recent years, mass spectrometry has been employed as a detection method.

Sample Preparation for Laboratory Mycotoxin Analysis

When performing analysis, it is important to choose the right sample preparation method to ensure accuracy, sensitivity of detection, repeatability and robustness, as well as fast sample preparation for high throughput. During laboratory analysis of mycotoxins, the sample preparation procedure typically includes extraction, purification and concentration steps.

Extraction of mycotoxins from samples is conducted by mixing the ground sample with the mixture of organic solvent and water, such as acetonitrile:water (80:20). Using methanol is not recommended, because it does not provide complete extraction. Prior to cleanup, the sample is filtered. Historically, mycotoxin analysis required extensive extract cleanup to minimize interference by matrix components. This holds true as new regulations continue to require lower detection limits.

Cleanup methodologies often include the use of phase extraction (SPE). Of the different types of SPE, one of the most common is the use of immunoaffinity sorbents that result in the selective retention and cleanup of mycotoxins. The drawback to using the immunoaffinity sorbents in the lab is that they are not compatible with the mycotoxin extraction solvent. In order to load the extract into the immunoaffinity SPE tube, the extract must be diluted with water, sometimes 20-fold, to prevent precipitation or folding of the protein-based antibodies by exposure to organic solvent. This presents an additional sample preparation challenge, as the grain extracts tend to form precipitates upon the addition of water and can clog the SPE columns. Thus, apart from the high cost of immunoaffinity SPE columns, the methods tend to be labor and timeintensive.

Super Tox SPE cartridges
Super Tox is a line of SPE cartridges for mycotoxin families that eliminates extra sample prep steps.

It would be beneficial to a laboratory to eliminate these extra sample preparation steps required by immunoaffinity SPE. Such cleanup SPE procedures are available and can be applied directly to the mycotoxin extracts without the need for further dilution, filtration and evaporation. A line of SPE cartridges for different mycotoxin families was recently introduced to the market. These SPE cartridges are compatible with the extracts generated during mycotoxin extractions and can be stored at room temperature. The tubes can also be used for cleanup of multiple classes of mycotoxins.

Analysis of Aflatoxins and Zearalenone

SPE cartridges are available for aflatoxins and zearalenone.
SPE cartridges are available for aflatoxins and zearalenone.

The following results employed SPE cartridges for mycotoxins that can be used for two aflatoxin classes, aflatoxins and zearalenone, and were applied to the cleanup of grain and peanut extracts. Results were compared to cleanup using immunoaffinity columns.

AflaZea SPE cartridges are based on the “interference removal” strategy that requires fewer processing steps compared to the “bind-and-elute” strategy of the other SPE. Peanut extracts contain not only co-extracted protein and complex carbohydrates but also fat. This extract was successfully cleaned using AflaZea SPE. When the SPE tube and a leading IAC column were applied to the peanut extract, both methods demonstrated good recoveries for spiked aflatoxins B1, B2, G1, G2 with AflaZea recovery values of 101–108% and immunoaffinity recovery values of 79–100%. However, the AflaZea provided better reproducibility for detection with a relative standard deviation (RSD) of 2–4% RSD versus 10–25% RSD with immunoaffinity SPE. This is likely because sample preparation using AflaZea is less tedious and takes one tenth of the time compared to immunoaffinity SPE.

Analysis of Deoxynivalenol

Wheat samples can be analyzed for deoxynivalenol using a new SPE cartridge.
Wheat samples can be analyzed for deoxynivalenol using a new SPE cartridge.

The following compares a new SPE cartridge for the analysis of DON, one of the Fusarium mycotoxins, with immunoaffinity SPE. Analysis of DON often is conducted using liquid chromatography (LC) with UV detection, so sample cleanliness is important to permit the separation of the DON peak from background interferences. The new SPE DON cartridge was compared to the immunoaffinity SPE for the cleanup and analysis of wheat samples. Clean chromatography and good recovery of spiked DON was obtained by both methods (86–97% RSD). However, clogging of the filters by the immunoaffinity SPE sample was observed during cleanup and complicated the sample preparation procedure. The SPE DON cartridge provided faster sample preparation.

Analysis of Patulin

Patulin is a mycotoxin commonly found in rotting apples.
Patulin is a mycotoxin commonly found in rotting apples.

Another SPE technology for mycotoxin analysis is based on molecularly imprinted polymers (MIPs), which are sometimes called “chemical antibodies” and mimic the performance of immunoaffinity sorbents. MIPs have binding sites that conform to the shape and functionality of specific compounds or compound classes. Strong binding of the analyte to the MIP makes it possible to perform intensive SPE washes that lead to very clean samples. Unlike immunoaffinity sorbents, MIPs are compatible with organic solvents and strong acids and bases.

Foods containing apples and similar fruits are required to be tested for patulin toxin, as they are the most common source for patulin exposure in humans. The MIP SPE procedure for patulin is faster than other SPE or liquid-liquid extraction methods and provides selective retention and superior cleanup. It is a robust method for analyzing apple juice and apple puree with HPLC-UV detection. After cleanup, patulin is quantified in apple puree at 10 ppb levels, which meet most regulatory requirements. The MIP SPE cleanup method eliminated 5-(hydroxymethyl)furfural (HMF) from the matrix, which sometimes appears as an interfering chromatographic peak when other sample prep methods are used. An SPE wash using sodium bicarbonate removed the interfering organic acids, while patulin was stabilized during elution at the end of the SPE procedure by using acidified solvent. Thus, most problems encountered during patulin analysis were resolved during this single SPE procedure.

Conclusion

As government regulations and consumer demand warrant cleaner, non-contaminated products, mycotoxin analysis will continue to be performed around the world. Careful selection of sample preparation methods is required for such analysis to achieve accurate testing results, best method performance and high laboratory throughput. Although many sample preparation methods exist, laboratories should choose the methods that not only provide adequately prepared samples, but also result in time and cost savings. The SPE technologies discussed in this article are sample preparation techniques that provide the required analytical sensitivity without capital expenditure into higher-end LC-MS equipment; the LC-UV and LC-FL methods can still be used. In addition, these SPE methods are simple, more robust, and less-time consuming compared to other SPE methods or liquid-liquid extraction.

All images courtesy of Sigma Aldrich