As toxic contaminants of very high concern, aflatoxins stand out as a key area of focus in today’s food safety testing arena. The issues surrounding the rising importance of accurate, defensible aflatoxin test results are numerous and complex. Chief among them are a growing public awareness of the health and economic implications of foodborne toxins, the increasing globalization of the agri-food supply chain, and the passage of stronger food safety laws in the United States, as well as in Europe, China, and India.
These developments are driving a surge in worldwide demand for food-testing services and prompting more frequent government and third-party assessments of laboratory performance against relevant national or international standards, such as the test performance requirements specified by the FDA, Codex Alimentarius, ISO, and AOAC. Test performance measures such as sensitivity, precision, recovery, and limits of detection and quantification play an essential role in determining the accuracy and reliability of a lab’s aflatoxin estimates and the acceptability of this data to regulators and clients.
Faced with burgeoning workloads and mounting pressure to verify the quality of their analytical results, laboratories entrusted with the mission of assessing the safety and marketability of agri-food products need to stretch their testing capabilities further than ever before. To equip laboratories for the current regulatory and economic environment, those capabilities must extend from obtaining unequivocal results across an expanding array of trade commodities and a broad range of country-specific maximum levels to empowering lean processes and faster turnarounds at a time when skilled staff appears likely to remain in short supply for the long term. This persistent workforce trend poses an undeniable barrier to operational excellence for laboratories in economically disadvantaged regions, where access to advanced scientific training is often limited, as well as in wealthier nations with graying populations.
Yet despite these challenges, there’s more reason than ever for food-testing labs to look to the future with confidence. When viewed with an eye to the radical innovations in testing technology that have recently come on the scene, the daunting stumbling blocks on the path to their goals shrink to manageable proportions, opening the way to meaningful improvements in data quality, operational efficiency, and client satisfaction.
Like most potentially game-changing opportunities, the chance to harness the transformative power of breakthrough technology comes with its own complications. In an aflatoxin test market packed with options that promise unprecedented benefits for food-testing laboratories and their clients, the attendant difficulties arise from the overwhelming abundance of possibilities: The more of these new applications there are to choose from, the more the distinctions between them start to blur, and the harder it may be to ascertain which one is best suited to meet the most urgent priorities of today’s food safety stakeholders.
The key to demystifying the relative merits of competing aflatoxin detection methods lies in a decision-making process informed by two essential sources of INSIGHT:
Aflatoxins, a family of toxins produced mainly by the common air- and soil-borne Aspergillus flavus and Aspergillus parasiticus molds, have been a focus of concern in the food safety community for more than 60 years. The potentially fatal toxicity of its most common and potent variant, AFB1, was first recognized in 1960 when aflatoxin-contaminated peanut meal was identified as the source of an disease outbreak that claimed the lives of more than 100,000 turkeys.
Over the subsequent decades, dramatic evidence that aflatoxin exposure poses a severe threat to human and animal health has continued to accumulate. Classified as a Group 1 carcinogen (i.e., a known cause of cancer in humans) by the International Agency for Research on Cancer (IARC) in 1987, AFB1 gained increasing notoriety as the subject of numerous cancer studies, including a widely cited quantitative risk assessment that estimated long-term exposure to low doses of the toxin may be responsible for as many as 25,200 to 155,000 cases of fatal liver cancer each year.1 A series of highly publicized fatalities from acute liver failure in humans and animals that ingested large doses of aflatoxins, including the deaths of more than 125 Kenyans in 20042 and over 110 pets in 2021,3 have likewise raised the impact of aflatoxins on global health to a top-of-mind public issue.
Other health risks linked to AFB1 exposure include chronic immunosuppression, impaired growth patterns in children, and kidney and neurological damage. Research on the combined toxicity of co-occurring mycotoxins suggests that the risk of these diseases and conditions may be heightened when food or feed contaminated with AFB1 contains additional Aspergillus metabolites, such as the three other main types of aflatoxin (i.e., AFB2, AFG1, and AFG2) and the aflatoxin precursor sterigmatocystin (STC).4,5
Although less potent than AFB1, its toxic metabolite AFM1, which ends up in the milk of animals that have consumed AFB1-contaminated feed, can cause serious harm in infants and young children, immature animals, immune-compromised individuals, and other populations whose less developed or weakened defenses against toxic invaders increase their vulnerability to the health damage done by aflatoxins.
At present, more than 100 countries around the globe limit the amount of aflatoxins allowed in food and feed to parts-per-billion (ppb) concentrations. Legislation on aflatoxins varies significantly in strictness and complexity from country to country, with European nations stipulating the most stringent and detailed regulations. These disparities in regulatory policies, which include different requirements for sampling and analytical methodologies and divergent data quality standards, as well as variations in legal limits, can complicate trade relations between importing and exporting nations, leading to disputes between trading partners and potentially hindering trade flows across borders. The exceptionally high bar for aflatoxin control set by EU member states and Japan, for example, may translate into market barriers for certain export products from countries with more permissive regulations (see Table 1).
Table 1: Maximum Levels (MLs) of Aflatoxins Permitted in Food and Feed in the U.S., EU, China, India, and Japan
To cultivate a strong and lasting market presence within tightly regulated trade zones, exporters in countries with less restrictive policies may need to overcome negative perceptions of their products in the minds of trade officials, importers, and consumers. Certification of agri-food products by accredited laboratories offers a potential solution to this dilemma. Solid evidence that a seller’s products comply with strict food safety standards set by widely recognized entities such as trade organizations, HACCP and HARPC schemes, or pre-export approval programs can help open the door to markets where aflatoxin restrictions are tighter than those of many exporting countries.
In the United States, for example, voluntary certification of a foreign supplier’s facility by an FDA-approved third party is one of the prerequisites for expedited market entry of food and feed imports. Imports are otherwise subject to a long and often arduous inspection process to ensure their compliance with all U.S. safety standards, including FDA aflatoxin limits. A general limit of 20 ppb total aflatoxins applies to food sold in the United States, with the exception of peanuts/ pistachios and milk, which are subject to limits of 15 ppb total aflatoxins and 0.50 ppb of AFM1, respectively. FDA limits for feed vary according to the age and category of the animal the product is intended for and range from 20 ppb for pets, dairy animals, and immature livestock to 300 ppb for finishing beef. (See Table 1.)
To benefit from reduced inspection levels in the EU, U.S. exporters must be able to document that their products satisfy far more exacting import requirements. Exporters of food grains, for example, must submit their products for testing prior to overseas shipment to certify compliance with EU aflatoxin limits as low as 4 ppb total aflatoxins (the sum of AFB1, AFB2, AFG1, and AFG2) and 2 ppb of AFB1. To get their shipments to market without risking long delays at customs and to fulfill the contractual terms of well-established EU importers, exporters of other important trade commodities, including tree nuts and compound and complete feed, likewise need documentary evidence of compliance with aflatoxin limits well below the maximum levels in force in the United States and many other major exporting regions. The trade revenues at stake are substantial. For instance, tree nuts, the most lucrative U.S. food export, reached a market value $3.1 billion USD in 2019, accounting for more than 26 percent of the total $11.7 billion USD in trade revenue from that year’s agri-food exports.6
For developing and transitional countries seeking a larger share of the U.S., European, and Japanese markets, ongoing compliance with import requirements represents a vital yet often elusive goal. Despite efforts to align their aflatoxin control measures with the standards of developed nations, some less developed countries may lack the requisite economic resources and technology tools to secure maximum competitive advantage in the world’s most profitable markets.
In recent years, the need for the kind of comprehensive, technology-driven food safety framework that supports consistent compliance with official aflatoxin control measures has become a major area of concern for emerging economic powers in the Asia Pacific region. The governments of China and India, the region’s top food exporters, have responded to this high-priority issue by increasing their investment in technology innovations that promise to bolster their competitive position in tightly regulated export markets and to protect public health and food security at home and abroad.
The current trend toward heightened concern about the public health and economic effects of aflatoxin contamination reflects the compelling realities of today’s global food and feed marketplace. In a broadening cross-section of countries, changing consumer demands continue to drive greater dependence on foreign suppliers. As agri-food producers, manufacturers, and retailers in higher-income regions and countries such as the North America, Europe, and Japan scramble to outcompete each other by lowering prices and bringing more diverse and out-of-season foods to market, they increasingly source raw ingredients and products from diverse geographies equipped with varying degrees of technological and testing prowess. At the same time, changing consumption patterns in developing countries, as well as in places with more developed economies such as the EU and Japan, are fueling demand for food imports from the United States. Chinese medicinal herbs and U.S. snack peanuts are just two examples of agricultural products that have given rise to thriving markets outside their countries of origin as a result of shifting consumer preferences.
These two economically significant export commodities fall along a wide spectrum of high-value staple and specialty crops that are particularly susceptible to infection by aflatoxigenic molds in the field and during storage and transportation. The high temperatures and extremes of water activity that promote aflatoxin biosynthesis in these molds are an endemic risk in steamy tropical and subtropical climate zones and in hot, drought-prone regions such as irrigated deserts. Aflatoxin-conducive environmental conditions prevail across much of the developing world, as well as in the southeastern and southwestern United States.
The onset of climate change has exacerbated these weather patterns in growing regions prone to aflatoxin contamination, increasing the incidence of outbreaks there and intensifying concern about the safety of imports from these areas. This situation has prompted more frequent inspections of aflatoxin-susceptible foods entering major importing countries and put their trade value in jeopardy, particularly in strictly regulated European markets. In 2019, failure to comply with legislated aflatoxin limits was the number one issue cited in hazard bulletins from the EU’s Rapid Alert System for Food and Feed (RASFF).7 These food safety alerts publicize violations of customs regulations across all member states, effectively banning noncompliant imports from all EU markets.
Growers and downstream supply chain partners in temperate climate zones also stand to lose from continued global warming. Aflatoxin-conducive weather patterns have already been linked to severe outbreaks of AFB1 in maize in 2003 and 2013 in southern and eastern Europe, areas where aflatoxin contamination had not previously been a major concern. The financial damage from the 2013 outbreak spread across food and feed supply chains in multiple European countries, extending downstream to the dairy sector in nations as far north as the Netherlands and Germany, where concern about the risk of AFM1 exposure resulted in market bans of milk from cows that had consumed the contaminated maize.8
Regardless of their sticker price, analytical tools that deliver erroneous or questionable test data can prove prohibitively expensive. The costs can range from error rates that put a laboratory’s accreditation status and reputation at risk to substantial financial losses for laboratory clients that bet the acceptance of their products in profitable target markets on faulty test results. The short-term hit to an exporting company’s bottom line can amount to thousands of dollars in expenses for warehousing, retesting, reprocessing, and shipping rejected commodities to less restrictive markets. The revenue loss incurred from selling noncompliant commodities at a steep discount exacts a still greater toll.
Over the long term, the economic fallout from a compliance lapse can extend far beyond the immediate cost of the violation. Future exports from the offending company, as well as those from other agricultural businesses located in the same region, may be subject to more frequent customs inspections and additional import restrictions.
From a trade standpoint, the worst case scenario arises from repeated cases of impounded and rejected shipments from countries in high-risk climate zones. Their costly impact can reverberate across the entire global food system, resulting in reduced trade revenues for exporting countries, diminished credibility with the importing countries and their consumers, gaps in the importing countries’ domestic food supply, and supply chain issues and lower incomes for local buyers and sellers in exporting and importing countries alike. Even more concerning is the potential public health threat from unsafe levels of aflatoxin contamination in food and feed that goes to market before the problem is detected.
The defining features of aflatoxin contamination further complicate the challenge of developing testing approaches that conform to widely accepted performance standards. In the absence of testing technology specifically designed to reduce the complications presented by these distinctive characteristics, the risk of this pervasive threat to the food chain evading accurate detection and measurement is too significant to ignore.
One of the hallmarks of aflatoxin contamination is its tendency to occur at extremely low levels in a broad range of agri-food commodities. Reliably determining ultra-trace levels of contaminants in food and feed samples is a notoriously difficult task, especially when testing compositionally complex commodities. Many of today’s most widely traded agricultural products, including traditional Chinese herbs, spices, roasted coffee, and peanut oil, fit this description. The exceptionally numerous and diverse constituents of these products typically possess chemical and physical properties that can interfere with the signal produced by an analytical instrument’s detector, compromising the accuracy of the test results. This physiochemical complexity can also hamper the sample extraction process, resulting in low yields of target analytes. These suboptimal recovery rates can cast doubt on the quality of data generated by the test and raise questions about whether the test procedures were performed properly. The need for testing approaches that address these issues is becoming increasingly acute as the range of agri-foods products subject to to trade regulations continues to broaden and those regulations tend toward ever greater stringency.
Another trademark feature of aflatoxin contamination that frequently undermines the accuracy and reliability of test result is the unevenness of its natural distribution pattern. Aflatoxin contamination generally occurs in widely scattered “hotspots” in bulk product lots. In practice, this tendency means the entire aflatoxin content of 20 metric ton lot of grain or peanuts may reside in a few isolated kernels, resulting in a substantial risk of obtaining wildly skewed aflatoxin readings from different portions of the same lot. Sample collection methods and sample preparation techniques that fail to compensate for this complicating factor are the two most common sources of inaccurate and inconsistent test results.
One of the most important advances in the quest for simple, practical approaches to accurately measuring aflatoxin concentrations in agri-food products emerged in the 1980s with the adaptation of immunoaffinity columns (IACs) to aflatoxin testing. AflaTest, the first IAC patented for this use, was introduced in 1986 by VICAM, a test developer based in Milford, Massachusetts. With the 1994 development of AOAC Official Method 99.31, AflaTest became the first IAC to serve as the basis of an AOAC-validated test method. Originally developed for aflatoxin determination in raw peanuts, peanut butter, and corn, this method has since been adapted for testing botanical roots. Validation by the USDA Federal Grain Inspection Services (FGIS), as well the test’s consistent conformance to all design and performance criteria laid down in the United States Grain Standard Act, further testify to the power of AflaTest to help laboratories and other operations that assess food safety build credibility with government agencies, clients, and the public.
The unique potential of this aflatoxin test kit to add value to food-testing services lies in its end-to-end approach to error reduction. Test protocols start laying the groundwork for success during the preliminary stages of sample handling. To reduce the rate of false negatives and false positives as well as the chances of variable results, the test instructions specify the use of sample collection and preparation techniques that enable users to capture a representative picture of the status of the lot under review. The recommended method starts with the creation a large aggregate sample comprising multiple incremental samples randomly drawn from every section of the lot. The materials are then ground in a mill and mixed together, so that any contaminated portions are evenly spread around. To ensure uniform aflatoxin distribution in the test sample, the next step calls for homogenizing a subsample of the ground materials in a blender with a solvent. (See Figure 1).
What ultimately distinguishes VICAM’s flagship product as a source of laboratory performance gains is its use of ultra sensitive and selective monoclonal antibodies to transform the once laborious, error-prone task of sample cleanup into a short, simple, foolproof process that generally achieves recovery rates of !90 percent. Located on stationary beads inside the column, the antibodies strongly bind with the target analytes and isolate them from the sample matrix. These aflatoxin-specific antibodies keep the target analytes firmly affixed to the beads while the matrix components are flushed out with water or a suitable wash buffer. To complete the process, the user adds methanol to the column to detach the aflatoxins from the antibodies and then collects and dilutes the eluate prior to injection into the detection system.
This cleanup approach requires no extra steps to concentrate the test sample or eliminate matrix components that might interfere with the accuracy of a test. Its simplicity offers the additional advantages of minimizing opportunities for human error while promoting fast, uninterrupted workflows. Instrumental analysis of the resulting sample delivers accurate, precise, reproducible readings of all four major aflatoxins, as well AFM1 and AFM2, at or below EU levels.
For applications that require the highest levels of sensitivity and precision, such as certifying shipments for export to the EU, the columns should be paired with an HPLC, UPLC, or LC-MS/ MS system. Combined with AflaTest column cleanup, UPLC analysis is sufficiently sensitive to quantify AFB1 and AFG1 without pre- or post-column derivatization to compensate for their weakened fluorescence, delivering accurate results, while significantly shrinking run times compared to conventional LC methods. The columns can also be used with a portable fluorometer for certification of food and feed for the U.S. domestic market and for high-efficiency laboratory prescreening and on-site quality control inspections.
VICAM has developed its antibody-driven cleanup approach to flexibly adapt to the unique characteristics of a diverse assortment of agri-food commodities while simplifying the complexity of the analytic task in the face of mounting regulatory and competitive pressures. The result of that development strategy is a growing line of aflatoxin test kits that incorporate significant innovations.
The introduction of a wide-bore (WB) column format, for instance, paved the way to major operational and productivity improvements. Their faster flow rate advances laboratory efficiency and budget goals, increasing throughput, saving time, and reducing the overall cost of testing. By accelerating sample cleanup, AflaTest WB columns enable laboratories to compress the time-to-results of HPLC, LC-MS/MS, and UPLC analysis, promoting prompt delivery of critical food safety data.
With the development of AflaTest WB SR, VICAM created a next-generation option for simultaneously improving workflows, analytic performance, and data quality. These multiple strategic benefits derive from a design that combines an important new feature with the proven strengths of the wide-bore format. While the expanded column width supports faster sample processing, the expanded packing volume of the antibody resin inside the column addresses the need for sound evidence of valid, accurate test results by significantly amplifying aflatoxin recoveries, particularly for AFG2.
The most recent addition to VICAM’s AflaTest line represents yet another revolutionary advance in the development of aflatoxin test applications that target the most vexing challenges confronting today’s food-testing laboratories. Specifically designed to overcome the difficulties involved in ultra-trace analysis of exceptionally complex samples, AflaTest WB SR+ offers the clear-cut advantages of an IA column that exceeds the exacting performance criteria specified by the AOAC. In addition to all the regulated aflatoxins, the SR+ column isolates and purifies the AFB1 precursor sterigmatocystin (STC), providing the basis for a more detailed risk profile of a product lot. Although not yet regulated worldwide, STC is a liver toxin and a Group 2B carcinogen (a possible cause of cancer in humans) that, as the compound that precedes the synthesis of AFB1, serves as an early warning sign of potential development of AFB1 contamination.
VICAM’s solution to the pressing analytic issue of low recoveries from problematic matrices draws its power from a new proprietary monoclonal antibody with exceptionally high affinity for aflatoxins and a robust tolerance to organic solvents. The new antibody’s super-strong bond with the aflatoxins in the sample resists disruption from matrix effects while its heavy-duty solvent tolerance allows for the use of high-strength organic solvent solutions, such as 40 percent methanol or 30 percent acetonitrile, during the sample preparation and dilution stages, without degrading test performance. At concentrations that high, a methanol or acetonitrile solution significantly increases extraction efficiency. This upsurge in both the amount of the target analyte extracted from the sample and the resistance of the affinity bond to matrix interferences in turn boosts recoveries to optimal levels of close to 100 percent.
The company’s Technical Services and Applications Manager, Nancy Zabe Collette, noted that in-house studies of AflaTest WB SR+ showed that it afforded recovery rates previously unobtainable with IA columns. “The new column yielded between 90 and 110 percent recoveries of aflatoxins from the traditional Chinese herb Fallopia multiflora,” she said. “Other commonly used methods of analyzing this herb typically yield much lower recoveries, in some cases as low as 30 percent.” The far-reaching potential of the column to improve the quality of the analytical result is borne out by its consistently superior performance with other highly complex samples. “When we tested its performance with nutmeg, chili pepper, roasted coffee, and cocoa,” said Collette, “the recovery rates of the four main aflatoxins were near or greater than 90 percent for all of the samples.”
In a comparative study of recoveries from a spiked solution, the usefulness of AflaTest WB SR+ for analytical challenges that defy conventional solutions proved equally clear. Compared to two other leading IA columns, the VICAM column obtained recoveries that were approximately 34 to 88 percent higher for AFG2 and approximately 9 to 52 percent higher for AFB2.
Collette sees the column’s versatility as another big plus. “The column promises to lighten laboratory workloads by enabling analysts to use the same simple procedures to test many different types of complex samples,” she said. “Instead of devoting a lot of time and effort to developing and validating a specific method for each one, analysts can repurpose a proven approach multiple times.” The method developed for Chinese herbs, for example, also delivers valid, accurate, consistent results for roasted coffee, cocoa, peanut oil, dog food, and a wide variety of spices.
Other standout capabilities of AflaTest WB SR+ further affirm its relevance as a multi-dimensional answer to the many complexities of aflatoxin testing. The column’s combination of outstanding sensitivity and specificity and expanded binding capacity make it an ideal tool for determining aflatoxins at either end of the measurement continuum, ensuring reliable quantitative detection across a range extending from 0.05 to 1,000 nanograms. Its ease-of-use enables lab techs to prepare high-quality test samples with minimal matrix interferences in a few simple steps. Designed for compatibility with several types of gold-standard instrumentation, the adaptable fast-flow, high-capacity column enables laboratories to fully capitalize on the advanced performance characteristics of their analytical instrument of choice. Paired with an HPLC, UPLC, or LC-MS/MS system, this high-powered cleanup column streamlines the path to comprehensive, definitive information about the aflatoxin content of samples that ordinarily resist fast, verifiably accurate analysis.
VICAM’s AflaTest product line is designed to optimize the results of an array of laboratory applications.
Zabe sums up AflaTest WB SR+ as a smart choice for laboratories striving to adapt to new, increasingly formidable demands. “The challenges of determining the safety and marketability of food in an era of expanding globalization, heightened regulatory control, and intensifying climate change calls for technology with the power to raise laboratory performance to the next level,” she said. “Combined with today’s most sophisticated analytic instruments, AflaTest WB SR+ equips laboratories with a technically elegant, UNIFIed approach to detecting and measuring all aflatoxins of concern, plus the AFB1 precursor sterigmatocystin, in a diverse range of complex samples. Unsurpassed for accuracy, sensitivity, specificity, and recovery of target analytes, the newest iteration of the AflaTest brand represents the next logical step in a test development strategy that marries industry-proven quality and reliability to boundary-breaking innovation.”