Food Exposomics and Chromatographic Strategies for Multi-Mycotoxin Detection in the Food Chain

Growing concern over the health effects of chemical exposure has intensified interest in exposomics, an approach that examines the totality of environmental exposures throughout life. Among the major exposure routes, food represents a critical source of chemical hazards, particularly mycotoxins, which are toxic fungal metabolites commonly found in cereals, nuts, fruits, spices, coffee, and animal-derived products. Climate change, global food trade, and the frequent co-occurrence of multiple mycotoxins are further increasing the complexity of food contamination and exposure assessment. Traditional risk assessment methods often fail to capture the combined effects of chemical mixtures and real-world co-exposure scenarios. In this context, advanced chromatographic and mass spectrometric techniques have become essential tools for food exposomics. Multi-mycotoxin “mega-methods,” combining high-resolution chromatography with sensitive detection platforms, enable simultaneous screening and quantification of regulated, emerging, masked, and modified mycotoxins across diverse food matrices and biological samples. These analytical approaches support both external exposome studies, focused on contaminated foods and environmental sources, and internal exposome investigations, involving biomarkers of exposure in biological fluids and tissues.

Research recently conducted highlighted current chromatographic strategies for comprehensive mycotoxin analysis within an exposomics framework, emphasizing their role in assessing co-exposure risks, identifying biomarkers, and improving understanding of exposure-related chronic diseases. By integrating food analysis with biomonitoring and omics technologies, exposomics-driven analytical chemistry is advancing food safety, toxicological research, and public health risk assessment. LCGC International spoke to Maykel Hernández Mesa and Ana Maria García-Campaña, corresponding authors of a paper published in TRAC Trends in Analytical Chemistry¹ based on this research, about their work.

How would you use liquid chromatography–mass spectrometry (LC–MS) to develop a multi-mycotoxin detection method in complex food matrices?

Developing a multi-mycotoxin detection method using LC–MS in complex food matrices (like cereals, coffee, tree nuts, dried fruits, spices, etc.) requires a systematic workflow. Because mycotoxins have diverse chemical structures and food matrices contain interfering components (fats, proteins, sugars), the method must balance broad extraction efficiency with high selectivity and sensitivity. Sample preparation is one of the most important steps. The goal is to extract a wide range of mycotoxins (polar to non-polar) while leaving behind as many matrix interferences as possible. One of the most popular applied method is the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) approach, using as extraction solvent a mixture of acetonitrile, water, and an acid (typically 0.1% to 1% formic or acetic acid), magnesium sulfate and sodium chloride to induce phase separation, and depending on the matrix complexity, dispersive Solid-Phase Extraction (d-SPE) with C18 or Z-Sep+ to remove fats or non-polar compounds, or PSA (primary secondary amine) to remove sugars/organic acids. For highly sensitive instruments, simply diluting the raw extract with the mobile phase could bypass tedious clean-up steps while drastically reducing matrix effects. In relation to the separation, a standard reverse-phase C18 column (often ultra-high performance, UHPLC, with sub-2mm particle sizes) is ideal for resolving the structural diversity of common mycotoxins (e.g., aflatoxins, trichothecenes, fumonisins, zearalenone, etc..), using as mobile phase a mixture of water with an ammonium modifier (e.g., 5 mM ammonium formate or ammonium acetate) and a weak acid (0.1% formic acid). The ammonium ions are vital for forming stable adducts for certain mycotoxins, like T-2 toxin. As organic solvent, acetonitrile or methanol (with 0.1% formic acid) are used. For detection and unequivocal confirmation, a Triple Quadrupole (QqQ) mass spectrometer operating in Multiple Reaction Monitoring (MRM) mode is preferred for its sensitivity and selectivity, using Electrospray Ionization (ESI). Because mycotoxins ionize differently, the method could utilize polarity switching. The most part of mycotoxins are analyzed by ESI+, but in some cases, ESI- can be used, sometimes analyzed as acetate adducts. For each target mycotoxin, the precursor ion and at least two product ions are selected with their specific collision energies (the most abundant ion, for quantification and the second one for structural verification). It is important to consider that complex food matrices frequently cause matrix effects (suppression or enhancement in the ESI source, altering signal intensity). To correct this effect, matrix-matched calibration must be applied prepared by spiking blank matrix extracts with mycotoxin standards, ensuring the calibrants experience the exact same matrix suppression as the unknowns. Finally, it is necessary to validate the optimized method according to official guidelines (e.g., SANTE, AOAC) by testing some parameters such as linearity, trueness (recovery, typically aiming for 70% to 120%), precision (repeatability and intermediate precision, RSD < 20%), limits of detection (LOD) and quantitation (LOQ), this last one, must be lower than maximum content allowed in different matrixes to ensure compliance with global regulatory limits.

What are the key challenges in separating structurally diverse mycotoxins (e.g., aflatoxins vs. fumonisins) using high performance liquid chromatography (HPLC)?

Separating structurally diverse mycotoxins simultaneously using HPLC is difficult because mycotoxins are not a single chemical class; they are a vast group of secondary metabolites of fungi with different physicochemical properties. When trying to separate a broad spectrum—like aflatoxins and fumonisins—in a single run, in traditional reversed-phase HPLC, one of the main drawbacks is the drastic polar difference. As example, fumonisins (like FB1) are highly polar and have very poor retention on standard C18 columns and tend to elute early near the dead volume, overlapping with polar matrix interferences. Conversely, aflatoxins (B1, B2, G1, G2) are relatively non-polar and hydrophobic aromatic structures that retain strongly. In this sense, it is necessary to develop a mobile phase gradient that is weak enough at the start to retain and resolve highly polar toxins (like deoxynivalenol or fumonisins), yet strong enough by the end to elute strongly hydrophobic toxins (like zearalenone or aflatoxins) without resulting in an excessively long run time. In this sense, these chemical structures dictate how the molecules behave under different pH levels, which can be considered in the mobile phase. In the case of fumonisins, which possess carboxylic acid groups and a primary amine, their ionization state changes dramatically with the pH. If the mobile phase is not adequately acidified, these groups deprotonate, making the molecule highly ionic, destroying its retention on a C18 column, and causing peak tailing. Aflatoxins, being neutral lactone rings, are largely unaffected by minor pH shifts, so it is necessary to find an optimum pH, using formic or acetic acid, that suppresses the ionization of acidic toxins to keep them retained, without degrading column stability or hindering the ionization efficiency required if the HPLC is coupled to a MS.

How do matrix effects influence quantification accuracy in food samples, and what strategies (e.g., internal standards, clean-up methods) can mitigate them?

Matrix effects primarily impact quantification accuracy through signal suppression or enhancement during the electrospray ionization process in LC–MS methods, which can lead to significant errors if the analytical environment of the sample differs from that of the calibration standards. To address this, one can take a physical approach by performing more intensive sample preparation, such as specialized clean-up steps, to remove interfering matrix compounds and make the sample behave more like a pure standard. However, in high-throughput environments like exposomics where extensive clean-up isn't always practical, matrix-matched calibration becomes the most common strategy to compensate for these effects. While this is widely effective, it does require a reliable source of blank matrix. For the highest level of accuracy, using internal standards with isotopically labeled compounds is the ideal solution, as they account for both matrix effects and recovery losses, though their high cost and limited availability often dictate the final choice. Ultimately, selecting the right mitigation strategy requires balancing the specific accuracy requirements of the application against the practical resources and time available in the laboratory.

In the context of exposomics, how would you design a “mega-method” using chromatography to simultaneously detect hundreds of chemicals in biological samples?

In this sense, it is important to differentiate between the ultimate goals of exposomics and the specific expertise a laboratory uses to tackle them. Exposomics pursues the characterization of the entire range of chemical hazards in biological samples, moving far beyond the limited scope of traditional human biomonitoring. This involves the ability to detect hundreds, or even thousands, of chemicals simultaneously. While generic non-targeted LC–HRMS approaches are the most suitable framework for this, they require extensive background in complex data processing and interpretation. Nevertheless, important chemical exposures can be overlooked as they are not successfully extracted from samples or satisfactorily ionized and detected by these generic analytical workflows, where too little attention is often paid to the sample-handling stages of the method. In our laboratory, our expertise has traditionally been built on developing targeted analytical methods for specific chemical families. While this might seem at the opposite end of the exposomics spectrum, it has provided us with a rigorous foundation in understanding sample preparation, as well as on matrix effects, chromatographic separation efficiency or MS fragmentation patterns. Therefore, our strategy involves enriching these high-quality methodologies with a broader range of compounds, following the work of pioneers like Rudolf Krska for multi-mycotoxin determination. Our goal is to transfer this rigor to a combined targeted and non-targeted framework to extend the scope of our analyses. Ultimately, a truly complete perspective requires more than a single 'mega-method'. It requires the strategic combination of multiple MS platforms and diverse chromatographic separations (reversed-phase, HILIC) or even capillary electrophoresis, to ensure no chemical class is left behind due to inadequate sample handling.

What are the advantages of coupling chromatography with high-resolution mass spectrometry (HRMS) for identifying “emerging” or unknown mycotoxins?

The primary advantage of coupling chromatography with high-resolution mass spectrometry (HRMS) over traditional low-resolution systems is the ability to perform non-targeted screening through accurate mass measurements. This level of precision allows us to determine elemental compositions with high certainty, which is essential for identifying emerging mycotoxins or unknown metabolites that aren't in our standard lists. Another transformative benefit is retrospective data processing; since HRMS typically operates in a full-scan acquisition mode, we capture a 'digital map' of the entire sample. This means if a new mycotoxin of interest is identified months after the initial analysis, we can simply re-interrogate the existing data without the need for physical sample re-analysis. Furthermore, HRMS enables us to obtain both precursor and fragmentation spectra (such as in Data-Independent Acquisition), which can be interrogated to look for structural similarities to known mycotoxins and decipher if modified mycotoxins not previously described are found. Nevertheless, it must be noted that not all are advantages, and some drawbacks must be considered, such as the time required for mass spectra interpretation or the complexity of those spectra when analyzing food or biological samples. For the first, the development of advanced software tools in combination with open available MS databases has been crucial to advance in this area. In the second case, the integration of ion mobility spectrometry (IMS) into LC–HRMS workflows is becoming increasingly relevant. While IMS is most widely known for its ability to resolve isomeric compounds, its most significant contribution in this context is often the mitigation of background chemical noise, which allows us to obtain much cleaner mass spectra for complex matrices.

Why are mycotoxins such as aflatoxins and Ochratoxin A considered major food safety concerns, and how do they enter the food chain?

Aflatoxins and Ochratoxin A (OTA) are among the most regulated and dangerous mycotoxins in the world. They represent critical food safety concerns because they are highly toxic, chemically resilient, and can contaminate a wide variety of foods. If crops are harvested with a high moisture content and not dried quickly enough, latent mold spores will rapidly proliferate and generate these mycotoxins. Aflatoxins, produced by Aspergillus flavus and Aspergillus parasiticus, include Aflatoxin B1 (AFB1), being classified in Group 1 as human carcinogen by the International Agency for Research on Cancer (IARC). It primarily attacks the liver, leading to hepatotoxicity, acute liver failure, and long-term hepatocellular carcinoma (liver cancer). OTA, produced by Aspergillus and Penicillium species is primarily a potent nephrotoxin. It exhibits also teratogenic, immunotoxic, and neurotoxic properties. Both kind of mycotoxins are stable molecules, even in food processing temperatures, including boiling, baking, roasting, and even pasteurization. These mycotoxins enter the food chain through a multi-stage fungal invasion process that can happen at any point from the farm field to the processed food. Cereals and tree nuts are highly susceptible to field contamination by aflatoxins. Also, when dairy cows consume feed contaminated with Aflatoxin B1, their livers metabolize it into Aflatoxin M1 (AFM1). This hydroxylated metabolite is then excreted directly into the cow's milk, arriving at dairy products consumed by humans, including infants. On the other hand, high humidity, poor ventilation, leaky roofs, and temperature fluctuations in silos or shipping containers create local areas where Penicillium and Aspergillus flourish, generating large amounts of OTA. Coffee beans, cocoa beans, dried fruits, spices, and cereal grains (wheat, barley) are frequently contaminated during storage. Also, OTA binds strongly to blood proteins and accumulates in the kidneys, liver, and muscle tissues of animals fed with contaminated grain, so, consuming these contaminated parts can introduces OTA into the human diet.

How does climate change influence the occurrence of mycotoxins in crops, and what implications does this have for food monitoring strategies?

Climate change is a relevant factor for mycotoxin contamination. Fungi are acutely sensitive to temperature, humidity, and plant stress, which are variables heavily disrupted by global climate changes. Aspergillus flavus, which produces highly carcinogenic aflatoxins, grows in hot, dry, subtropical climates. Historically, countries in northern and central Europe, as well as northern regions of the US, rarely worried about aflatoxins in local crops. However, rising average temperatures and prolonged summer droughts have caused Aspergillus to migrate northward. For example, countries like Italy, France, and parts of Central Europe now regularly face aflatoxin B1 outbreaks in local maize, leading to contamination of milk supplies via contaminated dairy feed (as aflatoxin M1). Changing climates mean plants are often hit with a drought early in the season followed by heavy rain later. This leads to multi-mycotoxin contamination, where a single crop contains a cocktail of multiple different toxins simultaneously.Climate change is forcing regulatory and industrial food monitoring to evolve. By integrating real-time weather data (temperature, humidity, rainfall during flowering/harvesting) with satellite crop health data, food safety agencies can map out "high-risk zones" each season and develop resources to detect contamination before the food even leaves the farm. Monitoring laboratories must select high-throughput LC-MS/MS methods capable of screening simultaneously a high number of mycotoxins in a single run. This is essential to capture the complex and changing profiles of co-occurring mycotoxins driven by climate change. In this sense, food safety authorities (like the EFSA or FDA) must continuously update their risk-assessment profiles for importing countries. It is important to consider that, due to the climate change, plant stress is increased, causing crops to metabolically alter mycotoxins as a defense mechanism—conjugating them to sugars (e.g., deoxynivalenol-3-glucoside) and producing “masked mycotoxins”. These masked mycotoxins are invisible to standard testing protocols but can be hydrolyzed back into their fully toxic forms in the human gut. Modern monitoring strategies should incorporate these modified forms into routine compliance limits.

What is meant by co-exposure to multiple mycotoxins, and why does it complicate traditional risk assessment models?

Co-exposure refers to the simultaneous ingestion of multiple mycotoxins, often from different chemical families, within the same food source or diet. Literature on mycotoxin occurrence consistently shows that samples are rarely contaminated by a single toxin; rather, they contain complex mixtures. This reality complicates traditional risk assessment models, which have historically focused on the toxicological effects of individual compounds in isolation. The main challenge is that these traditional models often overlook cocktail effects, such as synergism or potentiation, where the combined toxicity of multiple mycotoxins may exceed the sum of their individual effects. This can lead to an underestimation of risk in real-world exposure scenarios. Furthermore, the risk is often exacerbated by the presence of other co-contaminants such as pesticides, phytotoxins, or heavy metals in the same food source. Consequently, it is logical to move toward a more comprehensive characterization of exposure, addressing these chemical mixtures as a single relevant unit to better assess their cumulative impact on human health within a modern risk assessment framework.

How can the European Food Safety Authority (EFSA) contribute to regulate mycotoxins, and what limitations exist in current regulatory frameworks?

The EFSA plays a relevant role in protecting consumers from mycotoxin exposure across the EU. EFSA does not establish laws, but it provides the scientific background that the European Commission uses to propose strict and legal maximum limits for a safe consumption. At this respect, EFSA’s Panel on Contaminants in the Food Chain (CONTAM) evaluates extensive toxicological data to establish safe exposure thresholds. EFSA aggregates raw food consumption data from across all EU member states alongside millions of analytical data points collected from routine food testing. By modeling who eats what and combining it with known contamination levels, they pinpoint which populations (e.g., infants, toddlers, specific ethnic groups) are at the highest risk of exceeding safe intake thresholds. To ensure accuracy in enforcement, EFSA guides the harmonization of performance criteria for laboratory methods (such as LC–MS/MS) and standardized sampling protocols. This ensures that a batch of grain rejected at a port in Spain would be tested and rejected using identical scientific criteria at a border in Germany. While the EU has some of the strictest mycotoxin standards globally, several limitations could be considered in how these hazards are evaluated and managed. Once EFSA publishes a scientific opinion stating that a mycotoxin poses an urgent health threat, it can take months or even years of political negotiations, socioeconomic impact assessments, and industrial lobbying before the Commission formally translates that studies into a Maximum Limit (ML) in food law. Also, current EU regulations assess and enforce MLs for mycotoxins individually, but the real situation is that different toxins simultaneously occur in food and these combinations could have synergistic or additive toxicological effects. Another fact is the presence of masked and emerging mycotoxins (like enniatins, beauvericin, and moniliformin), that are frequently detected in food crops. While EFSA has acknowledged their presence in scientific opinions, many of these modified and emerging compounds still lack legal MLs because comprehensive toxicological data is scarce.

What role does exposomics play in understanding the link between dietary chemical exposure and chronic diseases?

Chemical exposomics represents a new scientific framework that goes beyond traditional epidemiological approaches. While traditional methods are typically hypothesis-driven, linking disease to a specific chemical, exposomics proposes a data-driven approach. From a global perspective, it pursues the definition of the ‘chemical exposome,’ encompassing all environmental chemical exposures from conception onward. This is achieved by measuring chemicals and their (bio)transformation products in biological matrices, known as the internal exposome, as well as characterizing exposure sources, or the external exposome. The aim is to establish a continuum from a chemical's source to its biological interaction, which acts as the initial molecular event. Subsequently, the goal is to link the sequence of biological events that occur from this molecular trigger to the manifestation of adverse health outcomes. In a dietary context, the use of high-throughput analytical platforms enables the holistic capture of all natural toxins, pesticides, environmental contaminants, and other chemicals to which humans are exposed, and their associated chemical burden, alongside their metabolic signatures. This holistic view is crucial for establishing more accurate links with chronic diseases, as it accounts for the cumulative and synergistic effects of multiple exposures that traditional methods often overlook.

Reference

1. Hernández-Mesa, R.; Alfonso Narváez, A.; M. Mar Delgado-Povedano, M. et al. The Role of Analytical Chemistry in Investigating Mycotoxins Within the Exposomics Framework. TrAC Trends Anal Chem 2026, 200, 118857. DOI: 10.1016/j.trac.2026.118857