Tag Archives: mycotoxins

Simon Hird
Ask The Expert

Plant Toxins – A Growing Global Food Safety Issue

By Simon Hird
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Simon Hird

Agriculture and food industries continue to be vulnerable to the complex problems of contamination with natural toxins. Mycotoxins, secondary metabolites produced by fungi, enter the food chain through infection of crops before or after harvest and are typically found in cereals, dried fruits, nuts and spices. Some have well established health impacts, both in humans and animals. A variety of testing solutions exist for mycotoxins, but growth in the use of methods based upon liquid chromatography with tandem mass spectrometry (LC-MS/MS) has enabled the determination of multiple mycotoxins. These methods are extremely sensitive and can be applied to the analysis of raw agricultural commodities, food ingredients and finished products.

Such LC-MS/MS techniques also serve as a powerful tool to investigate the presence of other natural toxins. They have been used for monitoring marine biotoxins[1], and to shed light on the distribution of toxins in terrestrial plants[2], highlighting them as a potentially serious food safety issue. Some terrestrial plants have evolved to produce secondary metabolites as defense mechanisms, which, while beneficial to the plant itself, cause harm to other organisms, including humans. In 2019, a humanitarian food aid product contaminated with tropane alkaloids (TAs) was distributed in Uganda, resulting in a foodborne outbreak which caused over 300 hospitalizations and five deaths[3].

Plant toxins can enter the food chain as constituents of plant products used in food processing or from the seeds and leaves of weeds mixed accidentally with the main food crop at harvest. Low levels of these toxins can be detected in cereals, herbal products, teas, salad crops and some animal products. One important class of plant toxins, pyrrolizidine alkaloids (PAs), are produced by a wide variety of plants commonly belonging to Asteraceae, Fabaceae and Boraginaceae families. Currently, there are more than 660 known PAs and metabolites. They are generally found in products such as honey, pollen, tea, herbal teas, food supplements, spices and aromatic herbs[4]. TAs are another class of plant toxins produced by plants, mostly within the Solanaceae family, and have been found in a range of agricultural cereal crops (e.g. linseed, soybean, millet, sunflower and buckwheat), tea, and herbal blends and infusions[5].

EU Legislation on Plant Toxins

The European Food Safety Authority (EFSA) has published the results of various risk assessments on those plant toxins considered to be the greatest risk to human health[6],[7], leading to the introduction of legislation on plant toxins in food by the European Commission[8]. Maximum levels have been set for PAs in herbs, spices, teas, herbal infusions and pollen products. These, which refer to the sum of 35 specified PAs (including their N-oxidized forms), vary between commodities. For example, the maximum level for PAs in most teas is 150 µg/kg, whereas the value for cumin is set at 400 µg/kg. Although there are more than 200 different TAs known, maximum levels have only been set for atropine and scopolamine (from 0.2 to 50 µg/kg, depending on the commodity). These regulations require that these plant toxins be monitored in specified foods by the Member State Food Safety Authorities and by food business operators, including those imported into the EU.

Access to data from retail surveys for PAs and TAs remains scarce when compared to that which is available for mycotoxins. However, in recent years, the number of food alerts reported on the Rapid Alert System for Food and Feed (RASFF) portal on the occurrence of PAs and TAs in different food products, exceeding maximum levels, has notably increased. The RASFF system was established to ensure the exchange of information between EU member countries to support swift reaction by food safety authorities in case of risks to public health resulting from issues with the food chain. Casado reported levels of PAs related to RASFF alerts with values ranging from 26 to 556,910 µg/kg[9], whereas the highest values of atropine and scopolamine were reported by Goncalves in tea and herbs (mean 173 and 147 µg/kg, respectively)[10]. In relation to consignments of cumin from Türkiye, a high rate of noncompliance with the relevant requirements provided for in EU legislation with respect to contamination by PAs was detected during official controls performed by the Member States[11]. The frequency of mandatory checks to be performed at border control has recently been increased to 30 %[12]. This has prompted greater awareness of the issue in other countries importing into the EU.

Techniques for Measuring Plant Toxins

Sampling plays a crucial part in precise determination of plant toxins levels in a certain lot, as contaminants within a lot may be heterogeneously distributed. It is also necessary to establish general method of analysis performance criteria to ensure that control laboratories use methods of analysis with comparable levels of performance. In December 2023, the European Commission published legislation establishing methods of sampling and analysis for the control of plant toxins levels in food[13].

Methods for PAs rely on extraction with acidified water, followed by solid-phase extraction (SPE) using a mixed-mode sorbent, which provides dual retention modes of reversed-phase and cation-exchange, followed by LC-MS/MS using alkaline or acidic chromatographic conditions[14]. The main analytical challenge is the presence of many isomers that are extremely difficult to resolve in the chromatographic dimension and exhibit the same MRM transitions. When attempting analysis in a single chromatographic run, one is left with a few pairs of coeluting isomers, which can be quantified as a sum. TAs are typically extracted with an acidified mixture of water and methanol/acetonitrile (including QuEChERS), followed by LC-MS/MS. Passing the extract through a simple ultrafiltration device or SPE cartridge can remove matrix co-extractives, enhancing method performance. To rationalize analyses in high-throughput laboratory environments, the scope of multi-mycotoxin methods can easily be extended to include the two regulated TAs, atropine and scopolamine[15].

While efforts have been made to address the food safety issue of plant toxins in Europe and reduce risk to the consumer, the number of food alerts seems to be on the rise. Fortunately, challenges with the determination of plant toxins in foods have largely been overcome, enabling testing to be carried out for checking regulatory compliance and monitoring occurrence, ensuring the safety of products for human consumption.

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References:

[1] Panda D. et al. (2022). Recent advancements in LC-MS based analysis of biotoxins: Present and future challenges. Mass Spec Rev. 41:766-803.

[2] Urugo, M. et al. (2023). Naturally Occurring Plant Food Toxicants and the Role of Food Processing Methods in Their Detoxification. Int. J. Food Sci. 2023 Article ID 9947841, 16pp.

[3] Abia W. et al. (2021). Tropane alkaloid contamination of agricultural commodities and food products in relation to consumer health: Learnings from the 2019 Uganda food aid outbreak. Compr. Rev. Food Sci. Food Saf. 20(1):501-525.

[4] Fuente-Ballesteros A. et al. (2024). Comprehensive overview of the analytical methods for determining

pyrrolizidine alkaloids and their derived oxides in foods. J. Food Compos. Anal. 125:105758.

[5] De Nijs, M. et al. (2023). Emerging Issues on Tropane Alkaloid Contamination of Food in Europe. Toxins 15(2):98.

[6] EFSA (2013). Scientific Opinion on tropane alkaloids in food and feed. EFSA Panel on Contaminants in the Food Chain. EFSA J. 11:3386.

[7] EFSA (2017). Risks for human health related to the presence of pyrrolizidine alkaloids in honey, tea, herbal infusions and food supplements. EFSA J. 15:4908.

[8] European Commission (2023). Commission Regulation (EU) 2023/915 of 25 April 2023 on maximum levels for certain contaminants in food and repealing Regulation (EC) No 1881/2006. OJ L 119:103–157.

[9] Casado, N. et al. (2022). The concerning food safety issue of pyrrolizidine alkaloids: An overview. Trends Food Sci. Technol. 120:123-139

[10] Gonzalez-Gómez L. et al. (2022). Occurrence and Chemistry of Tropane Alkaloids in Foods, with a Focus on Sample Analysis Methods: A Review on Recent Trends and Technological Advances. Foods 11:407.

[11] https://webgate.ec.europa.eu/rasff-window/screen/notification/651495

[12] European Commission (2024). Commission Implementing Regulation (EU) 2024/286 of 16 January 2024 amending Implementing Regulation (EU) 2019/1793 on the temporary increase of official controls and emergency measures governing the entry into the Union of certain goods from certain third countries. OJ L 2024/286.

[13] European Commission (2023). Commission Implementing Regulation (EU) 2023/2783 of 14 December 2023 laying down the methods of sampling and analysis for the control of the levels of plant toxins in food and repealing Regulation (EU) 2015/705. OJ L 2023/2783.

[14] Method Development and Validation for the Determination of Pyrrolizidine Alkaloids in a Range of Plant-Based Foods and Honey Using LC-MS/MS. Waters Application Note 720007624.

[15] Development of a Multi-Toxin UPLC-MS/MS Method for 50 Mycotoxins and Tropane Alkaloids in Cereal Commodities. Waters Application Note 720007476.

 

Apple Juice

Newly Discovered Fungus Helps Destroy Patulin Mycotoxin 

By Food Safety Tech Staff
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Apple Juice

Patulin is a harmful mycotoxin produced by fungi typically found in damaged fruits, including apples, pears and grapes. Recently, researchers from Japan identified a new filamentous fungal strain that can degrade patulin by transforming it into less toxic substances, which may lead to new ways of controlling patulin toxicity in food supplies.

Patulin is produced by several types of fungi, and is toxic to humans, mammals, plants and microorganisms. Symptoms of exposure include nausea, lung congestion, ulcers, intestinal hemorrhages, and even more serious outcomes, such as DNA damage, immunosuppression, and increased cancer risk. Environments lacking proper hygienic measures during food production are more susceptible to patulin contamination as many of these fungi species tend to grow on damaged or decaying fruits, specifically apples, and can contaminate apple products, such as apple sauce, apple juice, jams and ciders.

In the recent study, the research team from Tokyo University of Science (TUS) in Japan, screened soil for microorganisms that can potentially help keep patulin toxicity in check. The team cultured microorganisms from 510 soil samples in a patulin-rich environment, looking for those that would thrive in presence of the toxin. Next, in a second screening experiment, they used high-performance liquid chromatography (HPLC) to determine the survivors that were most effective in degrading patulin into other less harmful chemical substances. They identified a filamentous fungal (mold) strain, Acremonium sp. or “TUS-MM1,” belonging to the genera Acremonium, that fit the bill.

Patulin graphic
The mold TUS-MM1 can degrade the patulin mycotoxin.

The team then performed various experiments to shed light on the mechanisms by which TUS-MM1 degraded patulin. This involved incubating the mold strain in a patulin-rich solution and focusing on the substances that gradually appeared both inside and outside its cells in response to patulin over time.

One important finding was that TUS-MM1 cells transformed any absorbed patulin into desoxypatulinic acid, a compound much less toxic than patulin, by adding hydrogen atoms to it. Moreover, the team found that some of the compounds secreted by TUS-MM1 cells can also transform patulin into other molecules. By mixing patulin with the extracellular secretions of TUS-MM1 cells and using HPLC, they observed various degradation products generated from patulin. Experiments on E. coli bacterium cells revealed that these products are significantly less toxic than patulin itself. Through further chemical analyses, the team showed that the main agent responsible for patulin transformation outside the cells was a thermally stable but highly reactive compound with a low molecular weight.

“Elucidating the pathways via which microorganisms can degrade patulin would be helpful not only for increasing our understanding of the underlying mechanisms in nature but also for facilitating the application of these organisms in biocontrol efforts,” said Dr. Toshiki Furuya, Associate Professor at the Faculty of Science and Technology of the Department of Applied Biological Science at TUS and co-author of the study.

Sandra Eskin, OSU

Highlights from Food Safety Tech’s Hazards Conference

By Food Safety Tech Staff
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Sandra Eskin, OSU

The Food Safety Tech’s Hazards Conference + CFI Think Tank “Industry & Academia Advancing Food Safety Practices, Technology and Research” took place April 3-5 in Columbus, Ohio. The event offered two days of practical education on the detection, mitigation, control and regulation of key food hazards, followed by discussion geared toward identifying gaps for research and innovation.

Sandra Eskin, OSU

Sandra Eskin, Deputy Under Secretary for Food Safety, USDA FSIS, opened the program to discuss the agency’s proposed Salmonella in poultry framework. She highlighted the need for a more comprehensive approach that includes incentives to bring down the Salmonella load in birds entering the slaughterhouse, enhanced monitoring of safety measures within the facility, and enforceable product standards for raw poultry products.

Day one continued with a focus on Salmonella and Listeria. Barb Masters, VP of Regulatory Policy at Tyson Foods presented “Salmonella: What We’ve Learned and Remaining Gaps in Detection and Mitigation.” Masters highlighted key gaps in Salmonella detection, mitigation and research including:

  • Correlating what comes from the farm to what is entering a plant
  • Potential benefits of quantification testing
  • A better understanding of products that have the highest levels of Salmonella
  • Identification of virulence factors of different serotypes
  • The need for rapid testing methods that can be used at the plant level

Sanja Ilic, Ph.D., presented findings on the risks and most effective mitigation methods for listeria in hydroponic systems, followed by a session from Stacy Vernon, Ph.D., on recent listeria outbreaks in RTE meats and ice cream.

Shawn Stevens and Bill Marler

Attorneys Bill Marler, founder of Marler Clark, and Shawn Stevens of the Food Industry Counsel opened day two with an overview of the legal and financial risks of food safety hazards. The program continued with a focus on detection and mitigation of pathogens and biofilms.

 

Session Highlights

Application of Ozone for Decontamination of Fresh Produce with Al Baroudi, Ph.D., VP of Quality Assurance and Food Safety, The Cheesecake Factory, and Ahmed Yousef, Ph.D., Professor and Researcher with the Department of Food Science & Technology, OSU

Estimating Mycotoxin Exposure in Guatemala and Nigeria with Ariel Garsow, Ph.D., Food Safety Technical Specialist at the Global Alliance for Improved Nutrition (GAIN)

Mitigating the Risks of Salmonella and Listeria in Your Facility & Products with Sanjay Gummalla of the American Frozen Food Institute, and Rashmi Rani, Senior Manager of Food Safety and Quality Assurance, Schwan’s Foods

How to Use Whole Genome Sequencing in Operations To Improve Food Safety and Root Cause Analysis with Fabien Robert, Head of Zone AMS, Nestlé

Biofilm Prevention and Control Practices with Charles Giambrone, Food Safety Manager, Rochester Midland

On April 5, attendees joined the Ohio State University Center for Foodborne Illness Research and Prevention (CFI), founded and directed by Barbara Kowalcyk, for its annual “Think Tank.” The program featured student research presentations and an “Einstein Lunch” that brought members of industry together with graduate students and OSU researchers to identify gaps in research in the areas of pathogen detection and mitigation, handwashing and mycotoxins.

“We’re hoping this is the first of future collaborations with CFI and Food Safety Tech, where we have industry and academia presenting together,” said Rick Biros, founder of Food Safety Tech, the Food Safety Consortium and the Food Safety Tech Hazards Conference series. “This is something I feel both academia and industry benefit from, and I look forward to working with Barbara and CFI in the future.”

“I learned a lot myself, and it was great to see this program come together,” said Kowalcyk. “I want to thank the presenters, attendees and all the people who worked behind the scenes to make this event happen.”

Scenes from Food Safety Hazards Conference + CFI Thinktank

OSU 2023   OSU reception 2023  Sanja Ilic

Al Baroudi and Ahmed Yousef  CFI Think tank 2023  Saldesia OSU

Rick and Barbara Kowalcyk  OSu Reception - Steve Mandernach  Fabien Robert

 

 

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Food Safety Think Tank

Food Safety Tech Hazards + CFI Think Tank Coming to Ohio April 3-5

By Food Safety Tech Staff
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The Food Safety Tech’s Hazards Conference Series + CFI Think Tank, “Industry & Academia Advancing Food Safety Practices, Technology and Research,” will take place April 3-5, 2023, at Ohio State University in Columbus, Ohio.

The program brings together leading minds in industry, academia, standards and regulation to provide in-depth education and discussion on the most significant pathogenic and chemical risks facing the food industry today.

Building on the popularity of the Food Safety Tech Hazards virtual series, the in-person event will offer practical guidance and cutting-edge research on the detection, mitigation, control and regulation of the most significant foodborne illness risks.

The CFI Food Safety Think Tank on April 5 will bring food safety experts together to take a deeper look at the hazards discussed during the first two days of the conference. Participants will brainstorm in small groups to develop a roadmap on research, innovation, policy, and prevention measures that need to be taken to make our food supply safer in the future.

“Food safety hazards continue to be a challenge for all aspects of the food industry from farm to fork.” said Rick Biros, publisher of Food Safety Tech and director of the Food Safety Consortium conference and Food Safety Tech Hazards series. “The detection, mitigation and control of food safety hazards issues must be discussed among peers and best practices must be shared, something you can’t do virtually. The human connection is so important for conference attendees. Whether it’s a random connection over lunch, a one-on-one question with a speaker after a presentation or a seat next to a new friend in a learning session—connecting with others is what makes events so valuable. We are excited to bring this program, designed to help facilitate this much needed critical thinking and sharing of best practices, to OSU.”

Learn more and register here.

For sponsorship and exhibit inquiries, contact RJ Palermo, Director of Sales.

About Food Safety Tech

Food Safety Tech is a digital media community for food industry professionals interested in food safety and quality. We inform, educate and connect food manufacturers and processors, retail & food service, food laboratories, growers, suppliers and vendors, and regulatory agencies with original, in-depth features and reports, curated industry news and user-contributed content, and live and virtual events that offer knowledge, perspectives, strategies and resources to facilitate an environment that fosters safer food for consumers.

About Food Safety Tech Hazards

Launched in 2020, the Food Safety Tech Hazards series brings together industry leaders, researchers and regulators to provide in-depth education and discussion on the detection, mitigation, control and regulations of the most significant pathogenic and chemical risks facing the food industry today.

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.

Learn more about how to prevent and detect physical and chemical contamination risks in your facility at the Food Safety Tech Hazards Series: Physical and Chemical Contamination virtual conference. Now available on demand.

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.

Susanne Kuehne, Decernis
Food Fraud Quick Bites

Many Bad Apples Spoil the Bunch

By Susanne Kuehne
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Susanne Kuehne, Decernis
Rotten apples
Find records of fraud such as those discussed in this column and more in the Food Fraud Database. Image credit: Susanne Kuehne

Food fraud can have a substantial impact on a consumer’s health, like in this case of fruit juice that was sold (including to school lunch programs) in spite of contamination with arsenic and mycotoxins. The fruit used for the juice was decomposing, and also processed in a facility that unacceptably violated hygiene and food safety standards. The FDA filed a lawsuit against the company, which in the meantime has ceased operations.

Resource

  1. Vigdor, N. (November 10, 2020) “School Lunch Program Supplier Sold Juice With High Arsenic Levels, U.S. Says in Lawsuit”. The New York Times.

 

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