The Great Resignation began sweeping through U.S. workplaces in 2021, resulting in nearly 48 million workers quitting their jobs, according to an April 2022 article in Mashable. Surveys of workers revealed that their top reasons for leaving were better pay, improved benefits, a new career direction, or a better working environment. Nearly 30% of the U.S. workforce was impacted, and the trend continues into 2022 with no clear indication of when, or how, it might ease, according to 2022 research from Statista.

In addition, challenges involving supply chains, transportation, and price pressures are forcing food manufacturers to develop creative solutions that not only serve their immediate production needs but enable greater resiliency in the face of future challenges.

Food safety testing has often followed a predictable pattern: Regulatory, industry, and trade drivers may influence where and how testing takes place, but food manufacturers have long been proactive in developing strategic and tactical approaches to ensuring that food and beverages are nutritious and safe to consume. A closer look at the role that food safety holds across the food manufacturing life cycle can help identify areas in which small changes can significantly improve operational efficiency and worker satisfaction while maintaining the highest product quality and safety standards.

When a worker shortage and employee retention are hurting production as they are today, food processors may want to take a harder look at food safety testing technologies and methods that are easier on the bottom line and safer and easier for new workers to use.

Identifying Mycotoxin ­Contamination

Table 1. Mycotoxins commonly detected in food and agricultural products.

Produced by naturally occurring soil-borne molds, mycotoxins are highly toxic metabolites found in most field, orchard, and vine-grown crops (see Table 1). Heat stable and persistent, mycotoxins remain on crops after they’ve been harvested, stored, and processed. In fact, the United Nations Food and Agriculture Organization (FAO) has estimated that 25% of the world’s food crops are contaminated with mycotoxins. Recent studies suggest that contamination is more complex and involves the presence of multiple mycotoxins in a single raw material.

Aflatoxins are among the most widely known and highly regulated mycotoxins. Produced by Aspergillus flavus and A. parasiticus molds, aflatoxin B1 is classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC). Additional mycotoxins of food safety importance include fumonisin, ochratoxin A, patulin, ergot alkaloids, alternaria, deoxynivalenol (DON), nivalenol, zearalenone, and the combination of T-2 and HT-2. Each mycotoxin, or family of toxins, carries a unique toxicity profile, and regulatory guidelines are reflective of the intended use for the product. For example, the EU regulatory limit for aflatoxin M1 in milk products is 0.05 parts per billion (ppb); however, milk used to manufacture infant formula must follow a much stricter limit of 0.025 ppb.

The type or level of mycotoxin contamination varies with each crop season; therefore, having a process in place for screening can help identify high-risk raw materials, suppliers, and geographic regions. Severe weather patterns, warm and humid storage conditions, or even late crop planting may contribute to the severity of mycotoxin contamination.

Once a mold begins producing toxin, the contamination may remain highly ­localized to a very small area within a crop field or in a “hot spot” inside a storage bin. A single grain or nut kernel may constitute 100% of the aflatoxin contamination in each lot or shipment, for example, indicating the need for thorough inspection and careful sampling, especially at harvest.

Table 2. Lateral flow strip tests have come a long way and are highly sensitive, as these data from a 10-minute multi-toxin test procedure show.

In regions where environmental conditions (such as high heat or humidity) are favorable to mold growth, vigilance is key. Routine “upstream” monitoring is common, helping quality managers to identify and reject unsafe raw materials before they are allowed on site for storage or processing. Once mycotoxins enter the processing stream, the risks of cross contamination or further toxin production by the resident mold are always present. Food recalls or litigation due to mycotoxin contamination can be costly; the average recall costs the food industry between $5 and $10 million/incident, including insurance claims, legal representation, brand, and immediate and long-term business losses. The upstream detection of mycotoxins in raw materials also enables food manufacturers to find alternative markets for an ingredient that may not be suitable for their application but may be just fine for animal feed formulation.

Advancing Mycotoxin Testing Technologies

The Food Safety Modernization Act (FSMA) generated an upsurge in the use of rapid testing technologies. FSMA’s focus on ­prevention has enabled more food companies to better understand where mycotoxins come from and to manage the mycotoxin contamination of raw materials before they reach the processing facility. Early detection, combined with the unique challenges of our shifting workforce, creates the need for technologies that are simple enough to be used by staff with or without technical training or expertise. Adopting simpler test procedures that don’t require organic solvents and that are helped by automated data management are key factors that improve productivity, worker satisfaction, and safety, while giving the food manufacturer a leg up in meeting their own sustainability objectives.

Traditional mycotoxin testing methods are showing their age for a number of basic reasons. Some call for organic solvents, such as methanol, to extract toxins for analysis, which is what makes water-based test methods very attractive. Other methods, like ELISA, rely on employees handling the actual toxins and hand pipetting prior to sample analysis, risking exposure. Proper storage and disposal of unused testing supplies is also a consideration.

Fewer steps reduce error, bringing greater accuracy and better overall performance to screening tests.

As we know, not all mycotoxin testing takes place in the field. Sometimes it’s necessary to send samples for confirmatory testing to an analytical laboratory where trained lab technicians test for mycotoxins on analytical instrumentation including high performance liquid chromatography (HPLC), ultraperformance liquid chromatography (UPLC) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). These techniques can be automated to detect and quantify as many as one hundred mycotoxins in a single run. Effective onboarding and retention of new laboratory staff members may require investing in up-to-date instruments or methods, exploring service plans, or upgrading data handling software. Investments like these create an environment where employees are encouraged to learn, grow, work, and hopefully build a career.

Building for the future is always a good plan. There is an incredible opportunity amid the Great Resignation to pause and take a closer look at the technologies we use for food safety testing, and how they impact the employee experience. When our teams and the testing technologies they depend on work well together, food safety testing can deliver the most value.

Jackson is VICAM market development manager for Waters Corporation. Reach her at patricia_jackson@waters.com.

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Within the food industry, chemicals are routinely used to disinfect and sanitize food contact surfaces. These chemicals are necessary to ensure the continued safety of the food and drinks we consume—preventing microorganisms that could cause illness. There is a balance to strike; too little chemicals applied can result in inadequate efficacy, whilst too much can yield residues that do not meet regulatory standards.

Quaternary ammonium compounds (QACs) are surface-active substances often used as disinfectants. They are also used as biocides, pesticides, and as additives for technical applications.

QACs have the basic structure NR⁴⁺. Those possessing R groups with long alkyl chains are known to be especially effective as antimicrobial agents and particularly useful for the disinfection of containers and surfaces. This is especially relevant in the milk industry, as QACs are typically used to disinfect the insides of tanks used for transporting milk from farms to processing plants. If significant QAC residues are left behind after tank disinfection, allowing these compounds to leach into the milk and, ultimately, getting into the store-bought milk supplies at levels compromising personal health. Recent data points to nearly 12 percent of all monitored milk to be tainted with QACs. The primary QACs that may be found in milk are benzyldimethyldodecylammonium chloride (BAC 12), benzyldimethyltetradecylammonium chloride (BAC 14), benzyldimethylhexadecyl ammonium chloride (BAC 16), and didecyldimethylammonium chloride (DDAC). Their chemical structures and expected parent masses in solution are shown in Figure 1.

Figure 1: Chemical structures and expected masses of the four QACs analyzed.

Regarding safety and regulations, the European Union Reference Laboratory has taken the following position: “Because no specific maximum limit for residues of DDAC and BAC was established under EU Regulation No. 396/2005, the general residue limit of 0.01 mg/kg applies. In October 2012, the Standing Committee on the Food Chain and Animal Health (SCoFCAH) endorsed guidelines on measures to be taken regarding the presence of DDAC and BAC in or on food and feed. It was recommended that EU Member States carry out investigations on the possible causes of BAC/DDAC contamination and to put in place a monitoring program to get an overview of the BAC and DDAC levels in all food and feed of plant and animal origin. Considering that the current default MRLs for DDAC and BAC (of 0.01 mg/kg) are not a health standard, a temporary enforcement level of 0.5 mg/kg was agreed upon. As no specific residue definition was defined, there is still uncertainty as to how residues are to be expressed. Based on the first results of the monitoring program, a lower enforcement level for QACs is under discussion.”

The use of QACs is also regulated in the U.S. by the EPA under “Code of Federal Regulations (CFR)—40 CFR part 180. Once applied, the allowable residues and their subsequent monitoring are the responsibility of the U.S. FDA. Of course, the task of ensuring that the chemicals are prepared and applied properly avoiding inappropriate residues rests with the food processors.

With regulatory guidance in place, we pursued a LC-TOF (liquid chromatography time-of-flight) method for the analysis of the four most common QACs that may be found in milk. This technology takes advantage of the inherent mass accuracy and high resolution afforded by TOF detection for specificity and component identification.

As milk itself is a rather complex matrix (containing many components including proteins, fats, vitamins, and minerals), a further objective was to develop a rapid analysis method requiring relatively little sample preparation.

Path to Accurate Results
Standards and samples were analyzed using a PerkinElmer Flexar UHPLC System and AxION 2 TOF Mass Spectrometry detector. The analyzed product was a store-bought container of whole milk, spiked with standard solutions from 1 parts per million (ppm) to 0.05 ppm. Samples were injected after protein precipitation, centrifugation and filtration. Figures 2a and 2b show the chromatographic separation of the 0.5-ppm QAC standard, single injection and the replicates, separating the four QAC compounds in under 3.5 minutes.

Figure 2A: Chromatogram of 0.5-ppm QAC standard; EIC channels: BAC 12: 304.300; BAC 14: 332.332; BAC 16: 360.363; DDAC: 326.378.

Figure 2B: Overlaid chromatograms of 10 replicates of a preliminary QAC standard mix.

So how accurate are the measurements at the lower levels of detection? Figure 3 shows the calibration plot of BAC 14 over a concentration range of 0.05 to 1 ppm. A quadratic fit R² value of >0.999 demonstrates good linearity, assuring accuracy of results within the calibration range. Similar results were observed for the remaining three QACs.

Figure 3: Plots of 5-level calibration set for the BAC 14 (n=3 at each level).

The averaged MS spectra for all four QAC components are shown in Figure 4, highlighting the mass accuracy that was achieved using the integrated lock mass option. These were based on the expected exact masses for each component in solution.

Figure 4: Averaged MS spectra showing the mass accuracy achieved for each of the four QAC components.

The identity of the QACs was further confirmed with the help of elemental composition matching via AxION EC ID software. The accurate mass and isotope information for DDAC was simply entered into the software and searched against a selected database, in this case, PubChem. The search resulted in an elemental composition that perfectly matched DDAC.

Following a liquid-liquid extraction procedure, an extracted sample of whole milk and the same whole milk previously spiked with of 1-ppm QACs were analyzed. The overlaid chromatograms (EICS) of both extracts are shown in Figure 5. As shown in the expanded view, though trace levels of QACs were detected in the unspiked milk, none of them were above quantifiable limits.

Figure 5: EICs of 1 ppm-spiked whole milk extract (four large peaks) overlaid with that of the unspiked milk extract. Due to the very low levels, the EICs of the unspiked milk extract can only be seen in the expanded view.

Secured Milk Safety
With rising health concerns and the large quantities of milk that are consumed, it is imperative to have reliable procedures for the monitoring of possible unhealthy contaminants in dairy products. With this in mind, we have demonstrated the fast and effective chromatographic separation for the quantitative analysis of four QACs in milk by LC-TOF, with minimal sample preparation. The results exhibited exceptional reproducibility with more than adequate sensitivity for monitoring down to the current regulated levels in both the U.S. and in Europe. By using a TOF detector, the combination of averaged MS spectra, mass accuracy checks, and database search results allowed for the definitive identification and confirmation of the four QAC components.

In addition, such methodology has the benefits of offering the potential for horizon scanning. Not only are the compounds of interest identified and quantified, but as the whole mass spectrum of data is collected, any unexpected compounds can also be identified helping to prevent contamination scares before the milk is widely distributed.

Dr. Vosloo is the senior leader of strategy and global applications at PerkinElmer. Reach her at nicola.vosloo@perkinelmer.com. Reuter is senior LC and LC-MS strategic applications leader at PerkinElmer. He can be reached at wilhad.reuter@perkinelmer.com.

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The scope, relevance, and level of food safety regulation and testing have never been higher than in today’s global marketplace. President Barack Obama’s signing of the U.S. Food and Drug Administration (FDA) Food Safety Modernization Act (PL 111-353) in January signaled a renewed urgency and commitment to expanding the safety net of food testing and protection measures not only in the United States but around the world.

It’s no surprise. Each year, nearly 48 million people in the United States get sick from contaminated food; some 128,000 are hospitalized, and there are 3,000 foodborne illness-related deaths.¹ As more of our food comes from farther afield, the opportunity for contamination, both manmade and naturally occurring, can only increase. In the United States alone, an estimated 15% of the food supply is imported, including 50% of fresh fruits, 20% of fresh vegetables, and 80% of seafood.² The need for timely, accurate testing at every point along the farm-to-table pathway has never been more acute.

As a result, food suppliers, producers, manufacturers, and local, state, federal, and global regulatory agencies are all facing greater pressure than ever before to test more food products for more contaminants and quantify their presence at lower levels with greater accuracy—and in less time. Advances in screening technologies help to ensure that these critical lines of defense can meet the myriad of new testing requirements and, most importantly, protect the global food supply.

Advances in LC/MS

Food safety relies on a web of technologies to facilitate ongoing food supply monitoring. Mass spectrometry has a long and proven track record of enabling food testing labs to identify and quantify foreign substances in food. Recent advances in the integration of chromatographic separation techniques with tandem mass spectrometry bring a very high level of sensitivity and selectivity to food testing, particularly for the specific detection and identification of contaminants in the presence of other chemicals in a complex matrix.

In recent years, the utilization of liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) has grown rapidly and is now widely recognized as an ideal, highly specific, and extremely sensitive technique for testing food products with superior accuracy and higher throughput than other methods such as LC/ultraviolet (LC/UV), LC/fluorescence, or microbiological and enzyme-linked immunosorbent assay methods. The ability to monitor dozens, sometimes hundreds, of contaminants in a single run greatly improves throughput, and new levels of sensitivity allow more simplified sample preparation protocols. New levels of platform sensitivity allow detection of lower levels of contaminants with less sample preparation, and new workflows, such as multiple reaction monitoring (MRM³), provide unique specificity to help measure low level contaminants in complex matrices. These advanced LC/MS/MS platforms provide the capacity to screen for more analytes at lower levels, with greater accuracy, and in less time.

Recently developed workflows provide high sensitivity quantitative information, as well as supporting data to confirm the identification of contaminants found. The workflow uses the high sensitivity MRM functionality to provide sensitive and accurate quantitation while simultaneously acquiring full scan MS/MS spectra that can be matched to spectral libraries to provide high-confidence identification. The full scan MS/MS spectra functionality is also applied to screening for unexpected or unknown contaminants.

Simplified Sample Preparation

The drive toward decreasing time to results places significant demands on food testing laboratories to do more, faster. Preparative steps associated with sample testing are often the most time consuming and can be prone to error or the introduction of unrelated contaminants. As a result, for many technologies, sample preparation has been a rate-limiting step to achieving higher throughput in screening applications.

The sensitivity and specificity of LC/MS/MS can help to minimize extensive cleanup or pre-concentration steps. In some cases, a sample extract can be directly injected, enabling a “dilute and shoot” workflow.³ Also, sample derivatization is rarely required, unlike GC/MS, for which a time-consuming derivatization step is often necessary to improve ionization and/or volatility.

Great progress has been made toward applying more universal sample preparation to LC/MS/MS analysis. The QuEChERS (quick, easy, cheap, effective, and safe) extraction technique for extracting pesticides from food and feeds takes advantage of the sensitivity and specificity of LC/MS/MS for multi-residue pesticide screening and has the potential to be applied to a broader range of contaminants.⁴

Better Compound Coverage

Globalization of the food supply has necessitated screening for a wider range of potentially harmful substances. Raw materials and finished products alike are subject to contamination or adulterants that can originate almost anywhere along the farm-to-table pathway. Chemical testing has to cover a broader range of contaminants, and biological testing must identify bacteria and viruses that may be present in the food, as well as determine the exact serotype of each microbe, to locate the origin of the contamination.

Mycotoxins, for example, are low-molecular weight, chemically diverse toxins that are produced naturally by molds commonly found in grains and fruit and have been linked to a wide range of negative health effects. Aflatoxin, for example, is the most potent natural carcinogen known to man, and ochratoxin has been shown to damage the liver, kidney, and immune system.

LC/MS/MS is a powerful tool for the analysis of mycotoxins, because the workflow can screen for, quantitate, and identify several classes of mycotoxins in a single run, allowing laboratories to confirm the presence and structure of multiple mycotoxins and metabolites simultaneously in a significantly reduced amount of time. What used to require multiple analyses on multiple MS platforms now involves only a single LC/MS/MS run on a single system. This is not possible using technologies such as LC/UV, one of the most widely used screening techniques over the last 10 years.

In a recent multi-residue mycotoxin analysis and field study of wheat, barley, oats, rye, and maize grain, researchers at the University of Guelph, Laboratory Services Division, in Ontario, Canada were able to demonstrate a durable method for the simultaneous analysis of 22 mycotoxins in a single LC/MS/MS run using one common extraction technique rather than employing several different extractions and instruments.⁵ Further, the researchers found that “such a method offers a distinct advantage over other mycotoxin methods not only because it is cheaper and more efficient, but because it considers the toxicological relevance of the target mycotoxins.” Data generated from this study will also be used to advance the understanding of a wide range of mycotoxins, including cyclopiazonic acid, in cereals and grains and other matrices.

The FDA, which manages one of the world’s most sophisticated and far-reaching food supply monitoring programs, samples individual lots of domestically produced and imported foods and analyzes them for pesticide residues. LC/MS/MS gives the agency a technology that integrates quantitative and qualitative analysis on the same platform and performs automated identification of a wide range of contaminants simultaneously.⁶

Improved Technology

Developments in LC/MS/MS have been central to recent technology advances in food safety testing. LC/MS/MS enables laboratories not only to test for the presence of hard-to-detect toxins, but also to identify metabolites that may be formed after contamination and address the continually lowering detection limits required by global regulations. The technology is also widely applied in screening for other food contaminants such as pesticides, veterinary drug residues, marine biotoxins, and packaging materials.

Recent advances in mass spectrometry systems have significantly advanced both quantitative and qualitative analysis. The ability to quantify contaminants and obtain identification confirmation in a single run enables new levels of productivity. New levels of sensitivity and specificity allow lower levels of contaminants to be monitored and quantified with simplified sample preparation. Also, advances in high-resolution LC/MS/MS systems, complemented by new software tools, provide unique capabilities to screen for unexpected contaminants.

Outbreak investigations and routine screening play a central role in combating foodborne illness. Together, these efforts help identify new contaminants, new food vehicles, and unsuspected holes in the food safety net. They also help deepen the scientific understanding of how contamination occurs at specific points in the food supply chain, identify any likelihood that it may occur again, and plan ways to reduce or prevent it.

The number of components that must be monitored on the farm-to-table pathway will continue to increase, while allowable levels of contamination will continue to decrease. Food safety technologies will have to continue to advance their capacity to screen for more contaminants in less time and in easy-to-use formats. There will also be more requirements for general unknown screening or for identifying and measuring unexpected contaminants. This will require assays with high throughput, high sensitivity, and advanced tools to identify unknown or unexpected contaminants.

LC/MS/MS plays an important role in detecting threats to the food supply and in deepening our understanding of potentially harmful contaminants. As new technology advances are made, this versatile platform will continue play a greater role in protecting the global food supply.

Schreiber and Sims are technical marketing managers for food safety and environmental testing at AB Sciex. Reach Sims at (508) 383-7824.

References

1. United States Centers for Disease Control and Prevention. CDC estimates of foodborne illness in the United States: 2011 estimates. Available at: www.cdc.gov/foodborneburden/2011-foodborne-estimates.html. Accessed January 30, 2011.
2. United States Food and Drug Administration. Food Safety Modernization Act: putting the focus on prevention. Available at: www.foodsafety.gov/news/fsma.html. Accessed January 30, 2011.
3. Von Czapiewski K, Voller A, Schlutt B, Schreiber A. Tech note: simultaneous analysis of 14 mycotoxins and 163 pesticides in crude extracts of grain by LC-MS/MS. AB Sciex.
4. Anastassiades M, Lehotay SJ, Stajnbaher D, et al. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. J AOAC Int. 2003;86(2):412-431.
5. Martos PA, Thompson W, Diaz GJ. Multiresidue mycotoxin analysis in wheat, barley, oats, rye and maize grain by high-performance liquid chromatography-tandem mass spectrometry. World Mycotoxin J. 2010;3(3):205-223. Available at: http://wageningenacademic.metapress.com/content/nlv525p0123q1557. Accessed January 30, 2011.
6. AB SCIEX. Press Release: U.S. Food and Drug Administration selects AB SCIEX to provide mass spectrometry systems for food contaminant testing. October 20, 2010. Available at: www.absciex.com/Company/News-Room/US-Food-and-Drug-Administration-Selects-AB-SCIEX-to-Provide-Mass-Spectrometry-Systems-for-Food-Contaminant-Testing. Accessed January 30, 2011.

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