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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Food fraud is a significant concern for both consumers and producers. The scale of the problem is significant: 2016 research by Fera Science indicates that fraud accounts for up to 25% of all globally reported food safety incidents. Additionally, growing public demand for food authenticity means that consumers regularly pay a premium price for organic and sustainably produced goods, which is why unprincipled producers and distributors are flooding markets with adulterated, low quality, or mislabeled foodstuffs. This is not only damaging the livelihoods of legitimate businesses, but it’s also risking the health of consumers.

To make matters worse, the potential number of adulterants and the millions of different foodstuffs require a similarly wide range of test methods if food fraud is to be effectively detected and prevented. The rapid growth of global e-commerce also increasingly places food sales outside of regulatory oversight. To catch the food fraudsters, you first need to quickly and efficiently identify their handiwork, which requires special tools.

Assessing Food Authenticity

Analytical testing is an essential technology for assessing food authenticity, which is critical to protect the health of consumers, the food brand, and producer income. Testing is, therefore, a necessary part of an overall strategy to mitigate fraud risk. The techniques and reference databases used for authenticity testing are rapidly evolving, but more still needs to be done, not least in terms of consistency.

There is a lack of adequate testing and test uniformity across the globe. Additionally, many of the test methods reported in the literature either lack applicability to emerging frauds or are simply not deployed in an enforcement framework; however, in recent years, pressure has grown to improve traceability and accountability across the global supply chain, especially for the more commonly adulterated products.

Natural Sweeteners

Current demand for natural sweeteners is high. When consumers purchase a product, they want to be able to recognize the listed ingredients, and know that those ingredients are as natural as possible. This is one of the reasons for increased interest in honey, which has been a natural sweetener for thousands of years. Consumers want more of these natural sweeteners, so the production and sales of honey, particularly organic honey, are experiencing a hefty growth. We’re also seeing that consumers want natural product organic honey, called monofloral honey or unifloral honey, which is basically a honey that comes primarily from a specific type of flower. Consumers are willing to pay more for these products; therefore, we need to protect these consumers by making sure they get what they are paying for.

Creating a Buzz around Honey

One of the most widely adulterated products is the organic variety of honey, a high-value item prized for its unique properties. According to the U.S. Pharmacopeial Convention Food Fraud Database, it’s the third most targeted food for adulteration, after milk and olive oil. It’s also financially significant; a report by Grand View Research valued the global honey market at USD $9.21 billion in 2020 and expects it grow at a compound annual growth rate of 8.2%.

According to data from the United Nations Food and Agriculture Organization, China, Mexico, Russia, Turkey, and the United States are among the major honey-producing countries, accounting for approximately 55% of world production. The most common form of adulteration involves extending or diluting honey with other, less expensive sweeteners, such as corn, cane, and beet syrups. Any form of ingredient addition or substitution that creates a food safety hazard, such as the addition of an unlabeled allergen, must be addressed in the food safety plan.

Therefore, the ability to identify these substances quickly, efficiently, and consistently is essential to tackle fraudulent practices. What the food industry needs is analytical instruments and techniques that can consistently and rapidly fingerprint food and identify trace chemicals.

Setting the Standard

The good news is that liquid chromatography coupled with mass spectrometry (LC-MS) has emerged as the gold standard for analyzing trace constituents in food. The process enables food safety experts to map food components in an unprecedented fashion and will revolutionize how we manage and regulate the quality, safety, and authenticity of food.

While there has been work on developing ways to fingerprint foodstuffs, including honey, approaches among laboratories have varied in terms of sample preparation and analytical methods. There are also differences in terms of data processing. As a result, two laboratories analyzing the same sample could obtain slightly different results. To prevent the problems that may result from these variances, we should be looking at a standardized approach to fingerprinting and analysis.

Refining the Approach

Of course, we are trying to address two issues here: food safety and the quality and authenticity of the product. Each area is governed by separate sets of regulations. If we look at residues of contaminants in honey, such as pesticides, there also are differences between locations. For example, countries can have their own set of restrictions for the maximum limit for specific compounds. When we think about fingerprinting for honey, contaminants are a part of the picture, but the permitted levels vary between countries.

Food authenticity testing utilizing chemical fingerprinting strategies is emerging as a practical approach to tracking food fraud, as chemical fingerprints are virtually impossible to imitate due to their complexity. Regarded as the next-generation surveillance approach for chemicals in food, non-targeted analysis using high-resolution mass spectrometry coupled with innovative software enables the rapid characterization of thousands of chemicals in complex food matrices such as honey.

Currently, samples come from the field to the lab for testing; however, there is interest in potentially reversing this by bringing the lab out into the field. This interesting, but not yet recognized, capability would enable regulators and the food industry to rapidly respond more quickly to honey contamination—and to food fraud in general. By deploying the results of recent fingerprinting research in this way, we will be better equipped to protect consumers and producers alike.

A Global Perspective

The increasing globalization of our food supply chain raises the opportunity for food fraud. Experts predict that testing using methods such as those described above, will become more accessible, increasingly automated, and easier to perform. Fingerprinting methods—in which the entire molecular profile of a food can be obtained—will be a major feature of fraud prevention and identification systems in the future.

The good news is that current testing requirements have led to a rise in rapid, broad-coverage testing methods and technology to enable remote testing of food, in addition to improved testing within laboratory settings. Food testing laboratories can confidently measure contaminants that threaten the global food chain and supply and identify food fraud using these new approaches.

Dr. Bayen is an associate professor in the department of food science and agricultural chemistry at McGill University in Quebec, Canada. He is a recipient of an Agilent Thought Leader Award. Reach him at stephane.bayen@mcgill.ca.

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Image Credit: Bioo Scientific

Avian influenza is a major health and economic risk throughout the world. For example, the most notorious influenza epidemic, the Spanish Flu of 1918, caused the death of 20 to 50 million people worldwide. The avian influenza virus (bird flu) has been detected in six western states in the U.S., British Columbia, Asia, and Europe since the beginning of December. This disease is caused by certain strains of the influenza A virus, a type of highly infectious, negative sense, single-stranded RNA virus. Bird flu virus can be intermittently detected in chicken and turkey flocks in many parts of the world. These viruses can sometimes infect humans in food production sites and rapidly mutate to produce strains capable of causing global pandemics.

The World Health Organization (WHO) states that the primary risk factor for human transmission is exposure to live or dead poultry or contaminated environments in farms and animal markets. Additionally, the U.S. FDA says transmission is possible by exposure to environments contaminated by infected birds. It is transmitted by saliva, feces, and nasal secretions of infected birds. While the virus is generally located in the gastrointestinal and respiratory tracts of infected animals, it can also be found in the meat of infected birds. Avian influenza virus can also be located outside surface shell or within the interior of chicken eggs. According to the FDA, avian influenza is not transmissible by eating poultry or eggs that have been prepared properly and the chance of infected poultry or eggs entering the food chain is extremely low because of the rapid onset of symptoms as well as the safeguards in place. However, because of the devastating economic consequences of avian flu to poultry producers and the potential zoonotic hazards presented by exposure to infected birds, avian influenza is a major disease concern to poultry producers and human health officials alike. A great deal of time, effort, and expense is expended each year in curtailing the spread of avian influenza in chicken and turkey farms worldwide.

Individual strains of avian influenza viruses are classified according to the specific structures of two antigens, hemagglutinin (H) and neuraminidase (N), on the viral surface. These antigens control virus-host cell binding interactions during infection. Different virus strains possess different pathogenicity and zoonotic characteristics and can be classified into two different types: weakly pathogenic strains and highly pathogenic strains.

While weakly pathogenic strains of avian influenza can adversely affect poultry (chicken and turkey flocks), the more deadly highly pathogenic strains can cause tremendous economic damage to poultry farms and, more importantly, pose a direct threat to human health by occasionally crossing over the “species boundary” to infect and sicken humans. Highly pathogenic strains tend to be of type H5 and H7 and can rapidly infect an entire flock, killing 100 percent of the birds in less than 48 hours, according to WHO; infection can easily destroy an entire flock and threaten neighboring farms in the region. Some highly pathogenic strains, called zoonotic strains, can be transmitted to humans; these strains also usually possess the H5 or H7 subtype of hemagglutinin antigen. It is important to note however that some H5 and H7 avian influenza strains can possess low pathogenicity at first and gradually mutate to high pathogenicity forms. A minority of avian flu strains can be transmitted from birds to humans. These strains are most problematic since zoonotic strains, such as H5N1 and H7N9, can put farm workers’ health at risk and trigger global human influenza pandemics.

Measures to Detect and Control

The serious disease risks posed by poultry farming have created a need to prevent, detect, and control the spread of avian influenza throughout poultry farming regions and into the human food supply chain. The rapid mutation rates of the influenza virus make effective implementation of these control measures especially challenging.

Vaccination. Over the years, there have been numerous efforts to vaccinate poultry against bird flu to prevent infection or spreading of the disease. However, these programs have been generally unsuccessful and actually may hasten the evolution of more pathogenic influenza strains.

Improved sanitation and hygiene. Influenza is spread between birds through contact with feces as well as saliva and nasal secretions. Considerable reductions in the rate of spreading of influenza are achieved in farms and throughout neighboring farms through improved hygiene and feces handling techniques. For example, removing soiled shoes or boots after walking through influenza-contaminated areas can help to reduce the spread of the disease within a farm. Thorough cleaning and disinfection of contaminated areas has also been shown to be essential to limit the spread of the disease.

Detection/surveillance. There are a number of approved diagnostic techniques to detect the presence (and subtype) of influenza in bird flocks.

These methods include agar gel immunodiffusion, ELISA (enzyme-linked immunosorbent assay), AGP (alpha acid glycoprotein), ACIA (antigen capture immunoassays), and rtRT-PCR (real-time reverse transcriptase polymerase chain reaction). While a number of these diagnostic assays can be performed using “home brew” methods, a number of influenza assays are commercially-available including FluDetect, Binax, or Directigen.

Between 2006 and 2013, 569,000 flocks of broiler chickens in the U.S. were monitored by the USDA for the presence of either H5 or H7 avian influenza using these diagnostic tests; no samples from these flocks contained H5 or H7 type avian influenza virus.

Culling and quarantining of infected poultry. The adverse economic and human health consequences of the spread of avian influenza through poultry flocks creates a need for drastic measures; accordingly, the appearance of avian influenza in poultry flocks often necessitates the culling of infected birds in flocks and using quarantines to restrict the movement of eggs, poultry, and poultry products within infected areas. This very common practice has been shown to be highly effective at limiting the spread of bird flu in flocks and between farms. However, this practice tolls a heavy economic price on individual affected producers, in some cases all the birds in a large region are culled to eliminate bird flu from an agricultural region. In the case of persistent infections, culling and instating quarantines must be repeated over several years to completely eliminate the virus.

Antiviral drug treatments. Veterinary drugs are highly useful for the control of many diseases in livestock and poultry farms. For example, antimicrobial agents such as antibiotics and anticoccidal drugs are routinely used in poultry farms to combat a wide variety of diseases such as mycoplasmosis and coccidiosis. Several different antiviral agents, such as amantadine and ribavirin, have been shown to have strong anti-influenza virus activity. These drugs inhibit influenza virus replication at different points in the viral life cycle: Amantadine blocks the release of the virus into the cytoplasm from endocytotic vesicles by binding to the M2 channel; and ribavirin, on the other hand, is a nucleoside analogue which blocks viral RNA replication. Consequently, there was a great deal of initial interest in using antiviral drugs to control the spread of bird influenza.

Initially considered to be a useful facet of controlling avian influenza in poultry flocks, farmers sometime treat their flocks with antiviral medications to prevent and control avian influenza. Unfortunately, antiviral drugs are not effective at preventing or in controlling the spread of bird influenza in poultry flocks. It also has been shown to increase the mutation rate of the influenza virus towards higher pathogenicity and increased resistance to antiviral drugs. These changes have greatly reduced the therapeutic efficacy of these drugs in humans, and also created concern that poultry meat products will contain toxic residues of antiviral drugs. For these reasons, governments have banned the use of amantadine and ribavirin antivirals for veterinary use. Nevertheless, there are continued reports that these drugs are still being illegally used to combat bird flu in poultry farms and there is concern that residues from these drugs will adulterate the food supply. In fact, researchers have recently detected amantadine in chicken meat samples using liquid chromatography–mass spectrometry, or LC-MS. These residues pose a hazard to human health and have created great concern among international food safety regulatory agencies regarding the adulteration of poultry food products with antiviral drug residues. This concern has created an imminent need for methods to detect anti-influenza drug residues in poultry and egg products.

Antiviral Drug Detection in Meat

While several analytical methods have been developed to detect the amantadine and ribavirin antivirals, there are only two methods to detect these chemicals in poultry meat: LC-MS and immunoassay. While LC-MS methods work quite well for low-throughput meat sample testing applications, they have significant limitations that reduce their usefulness for routine and cost-effective sample analysis required to screen the world’s poultry meat and egg supply, these include:

• Expense and time: LCMS instrumentation can be expensive and operation requires considerable time for sample preparation and to perform the actual test;
• Throughput: Each instrument can only test one sample at a time; and
• Operator skill: A high degree of technical skill is required to perform LC-MS assay methods as well to perform maintenance on the instrument.

Immunoassays utilize the binding power and specificity of antibodies to overcome the practical limitations of LC-MS methods for the cost-effective detection of amantadine and ribavirin in chicken and turkey products.

Antivirals and Immunoassays

Immunoassays present many powerful benefits for the screening for antiviral drug residues in food samples. They are rapid and inexpensive to perform. ELISA immunoassays are used in microwell plates so many assays can be performed simultaneously. The highly specific nature of the detection minimizes the need for extensive sample preparation while allowing detection at very low levels similar to those achievable with LC-MS techniques.

Because of the recent use of amantadine to try to prevent bird flu in flocks and the danger it poses to the food supply, Bioo Scientific has developed an ELISA assay to detect amantadine in poultry products, including meat and eggs. The MaxSignal Amantadine ELISA Test Kit, with detection limits of 0.25 parts per billion (ppb) in poultry meat and 0.5 ppb in egg, is based on a competitive colorimetric ELISA assay. This kit incorporates a rapid sample preparation protocol for the extraction of amantadine from poultry samples. Amantadine residues present in the sample will compete for HRP-conjugated antibodies against amantadine, thereby preventing the amantadine-HRP from binding to the antibody attached to the well. The resulting color intensity, after addition of the HRP substrate (tetramethylbenzidine), has an inverse relationship with the concentration of amantadine residue in the sample. This assay can be completed in less than one hour. The kit is manufactured to the international quality standard ISO 9001:2008 (ISO CI#: SARA-2009-CA-0114-02-A).

In addition, Bioo Scientific is developing an ELISA for the detection of ribavirin in turkey and chicken food products. The ribavirin ELISA kit should be commercially available in the second quarter of 2015.

Dr. Krebs is director of Protein Chemistry and Engineering for Bioo Scientific. Reach him at jkrebs@biooscientific.com.

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Worldwide food safety concerns have risen dramatically as the number of food contamination incidents and product recalls has increased. Accurately monitoring contaminant levels in food and agricultural products is essential to assure the safety of the food supply and to manage human health risks.

It is well-known that the basic analytical requirements in food analysis are high-resolution, high-throughput, high-sensitivity detection and quantification of contaminants at or below the maximum residue limit (MRL) or tolerance of the compound in a given food matrix. Liquid chromatography-mass spectrometry (LC/MS) as the central enabling technology has been recognized as an indispensable tool in the food safety and quality control fields.1 LC/MS provides high-speed, high-resolution, and high-sensitivity separation of various chemical compounds.

Every food analysis starts with sample preparation, widely accepted as one of the most critical steps of LC/MS. Increased demand for higher throughput and accuracy and lower matrix interference from food analysis laboratories has made sample preparation the largest bottleneck. Currently, solvent extraction and solid phase extraction (SPE) are two of the most commonly used methods to isolate and/or enrich target analytes from food matrices. Done manually, these offline techniques are often labor-intensive, time-consuming, and costly, resulting in low sample throughput. Turbulent flow chromatography technology can do away with the need for lengthy offline sample preparation steps, thereby eliminating these disadvantages.

Turbulent Flow Chromatography

This article will review a number of key applications in food safety using turbulent flow chromatography. All experiments used an LC/MS system (Thermo Scientific Aria TLX liquid chromatography system) powered by turbulent flow chromatography (Thermo Scientific TurboFlow technology) to separate analytes from various matrices prior to MS/MS analysis. The system injected the sample directly onto a narrow diameter (0.5 mm or 1.0 mm) chromatography column (the patented TurboFlow column) packed with large particles. High linear velocities are created inside the column, which force large molecules to flow quickly through to waste while retaining the small molecule analytes.

The technology is an improvement over traditional SPE because it uses reusable extraction columns in a closed system, reducing the time required for offline sample preparation from hours to minutes. It also allows automatic removal of proteins and larger molecules in complex mixtures by combining turbulence, diffusion, and chemistry. Shows the typical configuration of a single-channel TLX system.

By injecting food samples directly into the LC/MS system, which eliminates time-consuming and costly sample pre-paration steps, food safety and quality laboratories can achieve significant analytical improvements. Turbulent flow chromatography technology also allows the broad selection of stationary phases. These features make the technology a versatile and important tool in the food safety arena.

Applications in Food Safety

1) Veterinary drugs and chemicals: In recent years, there has been increased concern about the use of unauthorized veterinary drugs and other potentially hazardous chemicals in farming operations. The U.S., Canada, the European Union, and many other countries have either banned or limited the usage of many veterinary drugs involved in food production.

Four common chemical residues in fish, shrimp, and pig liver were analyzed using a triple stage quadrupole mass spectrometer (Thermo Scientific TSQ Quantum Access).2 Malachite green (MG), a triphenylmethane dye, is an effective and inexpensive fungicide used in aquaculture, particularly in Asian countries. The principle metabolite, leucomalachite green (LMG), has been shown to accumulate in fatty fish tissues treated with MG due to its longer retention time.

Ciprofloxacin is a synthetic, broad-spectrum antibiotic belonging to the fluoroquinolone group that is used to treat severe bacterial infections. Tetracycline is a polyketide antibiotic that is highly effective against a number of gram-positive and gram-negative bacteria. The MRLs for these analytes range from 2 µg/kg for the sum of MG and LMG residues in fish muscle to 100 µg/kg for both ciprofloxacin and tetracycline in muscle for all food-producing species.

The total offline sample preparation time was approximately 30 to 40 minutes, including homogenization, centrifugation, and calibrator preparation. Compares representative standard high performance liquid chromatography (HPLC) and turbulent flow chromatography method chromatograms of 500 ng/kg (parts per trillion) tetracycline in a fish matrix. Using turbulent flow chromatography technology, the limits of quantitation (LOQs) were significantly lower (two- to 10-fold) for all four analytes using online extraction followed by LC/MS/MS compared to standard HPLC. This indicates that the LC/MS system can remove endogenous interferences, thus reducing ion suppression effects and improving detection limits.

2) Antibiotics in honey: Antibiotics are commonly used in beehives to control bacterial disease in honeybees, although caution is required to prevent persistent residues in food-grade honey. If antibiotic residues are present in high enough quantities, allergic reactions and bacterial resistance may develop. The conventional sample preparation for LC/MS/MS analysis of antibiotics in honey is time- and labor-intensive, often involving pH modification, hydrolysis, liquid-liquid extraction, SPE, solvent evaporation, and pre-concentration, and suffering, therefore, from low throughput. In addition, it is always an analytical challenge to deal with a large number of antibiotics belonging to different classes and often requiring multiple LC/MS methods.

Ten representative antibiotics used in honey, belonging to four different structural classes, were selected: sulfonamides, tetracyclines, aminoglycosides, and macrolides.3 The only offline sample preparation step required was the aqueous buffer dilution of raw honey to reduce the sample viscosity, which took less than 10 minutes. The online extraction clean-up was accomplished using a turbulent flow chromatography method involving two TurboFlow columns placed in tandem—a mixed mode anion exchange column and a polar-capped polymer-based column. Simple sugars were un-retained and moved to waste during the loading step while the analytes of interest were retained on the extraction column set. This was followed by organic elution to an end-capped silica-based mixed mode reversed-phase analytical column and gradient elution to a triple stage quadrupole mass spectrometer (Thermo Scientific TSQ Quantum Ultra). The total LC/MS/MS method run time was less than 18 minutes. A representative chromatogram of the 10 analytes at 100 ng/mL in 1:1 honey/ buffer was developed.

The results indicated that using two online turbulent flow chromatography extraction columns with different chem-istries extended the affinity range—further facilitating the separation and quantification of all of the representative compounds that have different chemical properties—in a complex honey matrix in a single analysis.

Quinolones, including fluoroquino-lones, in honey were also investigated.4 Instead of an SPE method, an online extraction method using turbulent flow chromatography was developed. The sample preparation time for the entire batch, including 16 compounds, dropped from five hours to 40 minutes, eliminating 80% of sample preparation time. The LOQs for the majority of analytes were 1 µg/kg (ppb) with no matrix interference. Representative selective reaction monitoring chromatograms at 20 µg/kg were developed and showed the selected ion transitions and retention times for the studied analytes.

3) Melamine in dairy products: The most notorious food safety incident in 2008 was the Chinese milk scandal involving melamine-tainted milk and infant formula. This incident triggered the largest global recall of Chinese-made diary products to date and prompted much stricter food safety regulations worldwide.

Consequently, scientists have developed many methods to analyze melamine residues in dairy-based products. Most of these approaches employ offline, disposable, cation exchange, SPE cartridges to prepare samples, coupled with LC/MS (MS/MS) analysis.

The goal in this experiment was to measure melamine with minimal sample preparation and high sample throughput. Using an LC/MS system, no offline sample extraction was required. Liquid or powdered dairy products were mixed with a solution of ammonium acetate in water and acetonitrile. After centrifugation, the supernatants were injected into the system. The quick-elute method took four minutes, with a one-minute data window. The lower detection limit was at least 50 ppb. Ion suppression caused by the co-elution of matrix components was minimal due to use of an atmospheric pressure chemical ionization source instead of the commonly used electrospray ionization source. The carryover level was also well-controlled and below 1%.5

Besides these representative studies, Thermo Fisher Scientific is actively studying an array of other contaminants (pesticides, mycotoxins, beta-agonists) in a variety of food matrices (fruit juice, wine, meat, and animal organs) using the TLX system with TurboFlow technology to minimize sample preparation, enhance analysis accuracy and reliability, and improve sample throughput.

Multiplexing capabilities of certain LC/MS systems such as the Thermo Scientific Transcend system allow up to four independent, parallel HPLC systems to run into a single MS, quadrupling the throughput of a traditional LC/MS. In addition, this unique multiplexing technology has attracted a great deal of interest because of its capability to run multiple methods simultaneously, one of the most important features desired by food safety and quality laboratories.

Online sample extraction utilizing turbulent flow chromatography coupled with LC/MS/MS and complementary techniques has gained popularity in the food safety arena. The objective of this technology is to provide automated, high-resolution, high-sensitivity, and high-specificity separation of target analytes from extremely complex food matrices, removing the need for manual sample preparation and increasing sample throughput. Turbulent flow chromatography also facilitates mass spectrometry detection and quantitative measurement and minimizes ion suppression and matrix effects. In addition, the multiplexing capability of the Aria TLX system can quadruple the throughput of a turbulent flow chromatography method, providing unmatched productivity and cost savings.

Dr. Shi is a senior scientist, Lafontaine is an applications chemist, Fink is a product manager, and Dr. Espourteille is manager, applications, at Thermo Fisher Scientific. For more information, contact Dr. Shi at yang.shi@thermofisher.com or at (508) 520-5575.

References

1. Soler C, Manes J, Pico Y. The role of the liquid chromatography-mass spectrometry in pesticide residue determination in food. CRC CR Rev Anal Chem. 2008;38(2):93-117.
2. Yang C, Ghosh D. LC-MS/MS analysis of malachite green, leucomalachite green, ciprofloxacin, and tetracycline in food samples using a TurboFlow method. Thermo Fisher Scientific Application Note 442. Available at: www.thermo.com/eThermo/CMA/PDFs/Articles/articlesFile_9061.pdf. Accessed August 2009.
3. Lafontaine C, Shi Y, Espourteille FA. Multi-class antibiotic screening of honey using online extraction with LC-MS/MS. Thermo Scientific Application Note 464. Available at: www.thermo.com/eThermo/CMA/PDFs/Articles/articlesFile_51570.pdf. Accessed August 4, 2009.
4. Hammel Y, Schoutsen F, Martins CPB. Analysis of (Fluoro)quinolones in honey with online sample extraction and LC-MS/MS. Thermo Fisher Scientific Application Note 465. Available at: www.thermo.com/eThermo/CMA/PDFs/Articles/articlesFile_51980.pdf. Accessed August 4, 2009.
5. Di Bussolo J, Rohm R. Comparing ESI and APCI sources to screen dairy-based foods for melamine by rapid on-line extraction with LC-MS/MS. Paper presented at: 57th ASMS Conference on Mass Spectrometry and Allied Topics; June 3, 2009; Philadelphia.

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