The post How High-Resolution Mass Spectrometry Can Increase Food Safety appeared first on Food Quality & Safety.

Pesticides are used extensively on a global scale to protect crops, ensuring they can be successfully grown, stored, and transported to meet consumer demands. The type of pesticide used varies widely depending on the produce in question, with insecticides, herbicides, rodenticides, and fungicides being the most common. A recent review by the Pesticide Action Network showed that there are more than 17,000 pesticide products currently on the market.

Solvent-based pesticides have traditionally been the pesticide of choice, but in light of growing health concerns, less toxic ionic pesticides are being more widely adopted. For example, glyphosate—an anionic pesticide—is now the most widely used pesticide in the world on GMO-engineered glyphosate-resistant crops. Recently, though, there has been growing public concern that any pesticide contamination in food could be a potential health risk, especially as pesticides can often remain in food at trace levels. This has resulted in increased attention from regulatory agencies and health researchers, who are seeking to better understand and monitor these residues.

To ensure that only minimal levels of pesticides are present in food, accurate quantification is required. Many methods exist for determination of pesticides, but gas chromatography (GC) and liquid chromatography (LC) combined with mass spectrometry (MS) are the standard techniques in regulatory test methods; however, these traditional analytical methods aren’t as effective for determining ionic pesticides, as the compounds are too polar to be retained and separated. In addition, it is difficult to maintain low baselines when analyzing ionic pesticides, making them an analytical headache. These challenges in current analytical approaches have been driving the need for more effective analytical techniques to continue protecting public health.

Taking Charge with IC-MS/MS

Ion chromatography coupled with tandem mass spectrometry (IC-MS/MS) can be used to effectively overcome the challenges faced by existing methods when it comes to anionic pesticide determination (see figure 1). Crucially, the technique is ideal for separating polar compounds and has been used to determine anionic polar pesticides such as glyphosate and glufosinate.

IC-MS/MS has a number of benefits that make it ideally suited for this application. The technique offers high selectivity and sensitivity, as tandem MS detection using selected reaction monitoring (SRM) eliminates sample matrix interference by only scanning for ions of interest. The method also provides low chemical noise, overcoming the baseline issue of GC-MS and LC-MS. With this technique, analytes are also provided in their ionic form, meaning electrospray can be used and the molecular ion retained. Further improvements in pesticide determination are enabled by the electrolytic suppressor, which neutralizes eluent and lowers the background while offering increased sensitivity for conductivity detection and improving the compatibility for MS.

Anionic samples are typically prepared for IC-MS/MS using the quick polar pesticides method (QuPPE) developed by the European Union Reference Laboratory for Pesticide Residues in Fruits and Vegetables (EURL-FV). This acidified methanol-based extraction method has been widely used and accepted for extraction of polar pesticides, according to a 2012 review published in the journal Analytical and Bioanalytical Chemistry, giving excellent results. IC-MS/MS used with QuPPE extraction provides a highly useful and sensitive approach for anionic pesticide determination, ultimately helping analytical scientists to better protect public health.

The Rise of Cationic Pesticides

Cationic quaternary amines, or quats, are a new class of ionic pesticide now gaining popularity. Unlike glyphosate, quats are permanently charged species, regardless of pH. Of these, paraquat, diquat, mepiquat, and chlormequat (see figure 2) are among the most important and commonly used.

Although ionic pesticides are generally less toxic than solvent-based ones, compounds such as paraquat and diquat are still highly toxic. Often, these pesticides are used late in the plant’s life as desiccants to kill the plant before the harvest. By doing this, farmers can bring the crops in earlier, before they are contaminated with mold during the rainy season. While this practice helps to guarantee the food supply, the late addition of these pesticides to the crop can cause problems as they can bind to the plant, creating a higher risk of food supply contamination.

The use of these cationic pesticides, and the risk of contamination, varies globally. Paraquat, for example, is a restricted-use pesticide in the U.S., and neither paraquat nor diquat are approved in the EU, but chlormequat and mepiquat are allowed. Alongside country-by-country restrictions on usage of different pesticides, the permissible quantities of these pesticides vary too. For chlormequat and mepiquat, the EU’s Maximum Residue Levels (MRLs) generally range from 0.01 – 0.05 mg/kg. These differences in approvals and MRLs mean that for products to meet the individual requirements of different countries, it is essential to be able to chromatographically resolve different ionic pesticides from each other to allow separate quantitation.

But to date, cationic polar pesticide analysis has lagged behind analysis of anionic pesticides, even by IC-MS/MS. Most notably, analysis is hindered by poor chromatographic resolution and high costs. While the permanent charge of quats makes them highly effective as pesticides, this feature also makes them highly impractical to derivatize for detection. Second, it also means they adhere, often irreversibly, to glass, metal surfaces, and particles such as clay. This leads to tricky sample preparation, and means chromatographic separation is not reproducible.

IC-MS/MS: A Powerful Quat ­Pesticide Determination Approach

With recent improvements in column stationary phases, IC-MS/MS can now be used to tackle challenging separations of cationic polar pesticides, including paraquat and diquat, which has been exceptionally difficult due to their similar structures and close m/z values for molecular and fragment ions (a difference of less than 2 a.m.u) (see figure 3).

Thanks to these advances, IC-MS/MS has been used to analyze quat pesticide levels in a range of some of the most widely consumed foods, showing promising results.

Here, we highlight two such studies: one examining cereals, and the other investigating wheat flour, baby food, and tea. In these experiments, the samples were prepared for analysis using QuPPE or adaptations of it. Overall, the IC-MS/MS method provided adequate resolution of the analytes of interest from the rest of the complex food matrix, giving more accurate results.

Cereals

Cereals are a principal component of many diets, yet the EU’s MRLs are much higher for pesticides in oat cereals. This is primarily because more pesticides are expected to be present in the produce due to higher levels used in cereal crop production. Matrix interference from complex samples makes it challenging to obtain accurate values, too, so this is factored into the MRL. With more analytical labs now using IC-MS/MS, these MRLs could be lowered in line with other produce, as matrix interference is reduced with IC-MS/MS.

Table 1. Summary of measured results and recoveries of added standard in cereal samples.

The study in question demonstrated that quaternary amine pesticides can be accurately and sensitively determined in oat cereals within 15 minutes using IC-MS/MS. Here, the sample extraction followed QuPPE and was passed through a Thermo Scientific Dionex IonPac CS21-Fast-4μm ion exchange column paired with a triple quadrupole mass detector. For determination of paraquat, diquat, mepiquat, and chlormequat, recoveries of 86% to 118% were obtained, and limits of detection (LODs) <0.1 μg/L or 0.5 μg/kg (see table 1).

Wheat Flour

Wheat flour is another dietary staple across the globe, and USDA estimates that 131.1 pounds of wheat flour was consumed in the U.S. per capita in 2019. Grain and grain products have particularly complex matrices, making samples challenging to prepare for determination. A simplified version of the QuPPE method has been used for the extraction of anionic polar pesticides, and this approach was also used for extracting cationic polar pesticides from wheat flour in the second study. Using IC-MS/MS here delivered excellent results, with apparent recoveries in QuPPE extracted wheat flour ranging from 97% to 113% (see table 2).

Table 2. Instrument apparent recoveries for four quaternary amine polar pesticides in wheat flour over a period of seven days.

Baby Food

MRLs in the EU for specific prohibited pesticides in baby food were previously set between 3–8 μg/kg; however, the European Food Safety Authority (EFSA) believes this may not be sufficiently protective for infants younger than 16 weeks of age. Yet, to date, there have been no further reductions to MRLs, as suitable analytical methods for detection with improved sensitivity are scarcely used.

IC-MS/MS can be used for effective determination of quaternary pesticides in baby food, though. Following the approach used with wheat flour (in the same study), the simplified QuPPE method can be used to prepare samples and extract pesticides from carrot baby food. The IC-MS/MS method, using the Dionex IonPac CS21-Fast-4μm ion exchange column paired with a triple quadrupole mass detector, worked extremely well, giving apparent recoveries of the pesticides ranging from 96% to 103% (see table 3).

Table 3. Instrument apparent recoveries for four quaternary amine polar pesticides in carrot food over a period of seven days.

Tea

Testing and regulation of beverages have also greatly increased over recent years. One of the most widely consumed beverages—tea—can suffer from pesticide contamination; that is, pesticides that remain in tea leaves can leach into the drink when hot water is added. Determination of these compounds is therefore essential.

To show the versatility of the IC-MS/MS method, tea infusions from both green tea and white tea were prepared as part of the second study and filtered for analysis by IC-MS/MS. The method effectively separated the four common quat pesticides, and corrected apparent recoveries were 94% to 102% for green tea and 92% to 106% for white tea (see table 4).

Table 4. Instrument apparent recoveries for four quaternary amine polar pesticides in green tea and white tea over a period of seven days.

Paving the Way for Food Safety

Quaternary ionic pesticide use is growing, which is bringing many advantages to food production and distribution. While cationic compounds have typically proved difficult to analyze, advances in ion chromatography column technology are now enabling IC-MS/MS methods that can accurately and sensitively determine them while significantly simplifying analysis.

Eventually, as such IC-MS/MS approaches continue to gain traction for the analysis of quats, the possibility opens for MRLs to be lowered. These lower MRLs will drive improved agricultural practices, alleviating concerns for consumers and regulators and ultimately improving the protection of human health.

Man is product marketing manager specializing in ion chromatography at Thermo Fisher Scientific. Reach her at wai-chi.man@thermofisher.com.

The post Improve Food Safety with Cationic Polar Pesticide Determination appeared first on Food Quality & Safety.

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.

The post How to Simplify Mycotoxin Testing in the Food Industry appeared first on Food Quality & Safety.

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.

The post How to Ensure Honey Purity through Mass Spectrometry appeared first on Food Quality & Safety.

Food fraud has become a topic of major concern over the past decade, primarily due to major incidents, such as the 2008 Chinese melamine scandal and the 2013 European horse meat scandal, and subsequent increased customer awareness and media coverage.

The cost of food crime is considerable. This expense has helped refocus attention on developing measures to ensure the integrity of the food supply chain, with an increase in demand for food fraud detection to be proactive, rapid, and reliable to maintain the security of the food chain while also acting as a deterrent.

There are many types of food fraud, including:

• Substitution of part or all the food with a lower value commodity;
• Addition of a component to increase the value of the overall product; and
• False claims on product labels that increase their value, such as “organic,” “welfare friendly,” “fair trade,” or “country of origin.”

Direct Analysis Using Mass Spectrometry (Direct MS)

The primary objective of authenticity testing is rapid verification, from raw ingredients through to finished (processed) products, to support traceability systems. Development of technologies that can be used to rapidly differentiate authentic products from fraudulent ones represents a significant challenge. One approach is the direct analysis of samples using mass spectrometry (MS) without any pre-treatment (e.g., extraction or chromatography). MS generated using various types of ambient ionization are used to create multivariate statistical models. Most applications have used linear discriminant analysis on principal component analysis (PCA-LDA) reduced data for the generation of predictive models, but others have explored machine learning approaches.

The result of the subsequent sample classification is presented and refreshed in real time. In all cases, validation is essential to evaluate the accuracy of the models. Analysis of a sample and the generation of results takes only a few seconds, enabling faster decisions and support for next steps. Let’s look at a few examples.

Rapid Evaporative Ionization Mass Spectrometry (REIMS)

REIMS allows for the collection of mass spectrometric data directly from the surface of biological samples, without any sample preparation. The technique was originally demonstrated to show promise for detection of cancerous tissues during surgery but has subsequently been used for investigation into food and beverage fraud, especially in the seafood and meat sectors. This work is conducted on a high-resolution instrument, the quadrupole time-of-flight (Q-TOF) mass spectrometer, to ensure enough selectivity to differentiate components and increase the specificity of the statistical model. REIMS typically uses a surgical diathermy sampling device, the iKnife, but there is growing interest in alternative means to generate the aerosol from the sample, such as other designs of monopolar probes, bipolar forceps, and use of lasers.

When it comes to fish and shellfish, we often don’t get what we ask for. Fraud is common. For example, one can get high-quality salmon substituted with lower quality salmon species, wild swapped for farmed, and, in some countries, rainbow trout is often mislabelled and sold as salmon. Typically, polymerase chain reaction (PCR) methods are used, which exploit minor differences in DNA sequence between different fish species. A small piece of fish DNA is copied many times using PCR and compared with a large, authenticated database of fish species using matcher software to ensure accurate fish species identification.

However, such techniques comprise multiple steps and can take hours. REIMS offers an accurate, high-throughput, cost-effective alternative to screen large numbers of samples for discrimination among fish species. After construction and validation of well-established models, the identity of blind fish fillets can be given in real time without any sample preparation. It has been demonstrated that REIMS can be applied as a rapid screening technique to detect various species of white fish, salmon, tuna, and other sea creatures, to complement existing DNA methods. In addition, there is some evidence that the same approach can be used to monitor the quality of products, such as shelf life and degree of lipid oxidation of fish oils during storage and in real time during cooking.

Since high quality meat demands premium prices, producers of meat-based products might be tempted to blend these products with lower cost meat, cuts from the same animal, or other bulking agents. Moreover, the labelled meat contents may no longer be met. All three types of adulteration are difficult to detect in processed products and lead to deterioration of product quality. REIMS has successfully been used to measure meat quality, fraud, and safety, including determination of species, country of origin, and substitution with cheaper cuts of meat.

Although REIMS is rapid and simple to use, the technology is coupled to a high-resolution mass spectrometer (HRMS), which may prove prohibitive for most point-of-control testing. REIMS has been installed and used effectively in an abattoir to detect boar taint, demonstrating that this is a technology that has practical potential to be used closer to the points of production and control if the costs can be reduced. However, there are innovative solutions being explored on the potential of other ambient ionization techniques, but fitted to a compact, easy-to-use nominal mass detector, which has greater potential for deployment away from the research laboratory environment.

Atmospheric Solids Analysis Probe (ASAP)

There are other types of ambient ionisation, for example, ASAP, which has recently been interfaced to a much simpler mass detector to provide a new, low-cost, dedicated direct analysis MS system. The sample or a related solution is simply applied to the glass capillary probe of the ASAP under controlled heating without any significant sample preparation. Upon thermal desorption at high temperatures, the vaporized molecules are ionized at ambient pressure before entering the mass spectrometer.

Although the MS-generated results from the ASAP are not the same as REIMS and comprise ions from a lower molecular mass range, there remains enough information to generate reliable models. As a proof of concept, the system has been used to generate multivariate statistical models for the detection of substitution fraud in dried oregano. The results from the validation study demonstrate the capability of the solution as an accurate, robust, and routine screening tool for the real-time recognition of adulteration in herbs. There are also investigations underway looking at the performance of this dedicated, compact, direct MS platform for other applications, including cocoa butter quality control, detection of fraud in edible oils, and mislabelling of honey. The data from this simple mass detector, when combined with multivariate statistics, proved able to rapidly differentiate sample types with good accuracy.

Direct MS enables rapid discrimination and classification of different raw ingredients and foods or feed using multivariate statistical reference models to quickly screen samples for signs of fraud. The absence of any sample preparation of chromatography steps when combined with a simple results dashboard makes results possible in seconds, thus speeding up decision making. It can be used for classification, to give immediate yes/no decision making, so the technique is of interest for point-of-control testing, bringing the analysis closer to the point of production, or at critical points in supply chain. There is a growing range of applications across food, meat, and crop sciences.

Dr. Hird is a principal scientist at Waters Corporation in the U.K. Reach him at simon_hird@waters.com.

The post Using Direct Mass Spectrometry to Verify Product Authenticity appeared first on Food Quality & Safety.

Accurately detecting pesticides in food is critical to your laboratory’s success and the safety of consumers. At the same time, analyzing pesticides in food is no easy task. There are multiple pesticide residues belonging to multiple classes. This e-book showcases a combination of LC-MS/MS and GC-MS/MS to deliver fast quantitative analysis of 1000+ pesticides in food. With these systems, you get the high sensitivity you need to generate high-quality data for complex samples, quickly and without compromising data quality.

Click here to read the details.

The post Fast, Quantitative Analysis of 1000+ Pesticides Using MRM Mass Spectrometry appeared first on Food Quality & Safety.

Targeted pesticide residue analysis is a well-established and essential part of modern food testing. Laboratories routinely determine pesticides present in food samples against a specific target list to ensure food products comply with the maximum residue levels (MRLs) set by governments (European Commission) and national food safety authorities.

Growing concerns over food safety and expanding international trade have led to the development and enforcement of stricter pesticide regulations in recent years. In 2014, China’s Ministry of Agriculture and Ministry of Health jointly issued a revised national food safety standard, which expanded the number of categories of pesticide residues and total number of MRLs. Together with the Japanese Positive List System and European Union (EU) Directive No 752/2014, these standards are amongst the strictest food safety regulations in the world.

Traditionally, separation technologies such as gas chromatography (GC) and liquid chromatography (LC), coupled with triple quadrupole (QqQ) mass spectrometry (MS), have formed the mainstay of targeted pesticide residue quantitation workflows. The high sensitivity and selectivity offered by these QqQ-based techniques allow analysts to confidently identify and quantify even trace levels of known contaminants, while their robustness ensures fast, reliable, and cost-effective routine analysis.

Yet new pesticides are continually being developed and applied to crops around the world. The growing complexity of global food supply chains means that pesticides approved for use in one country can unexpectedly end up in food consumed in another, where the pesticides are not approved. Other chemicals, previously undetected in food samples and not on target lists, can also enter food chains during product preparation, transport, and storage from an often-surprising range of sources.

As a result, food safety laboratories are not only faced with an increasing number of analytes to screen for—they must be vigilant for new chemicals too. Of course, all of this must be achieved with high turnaround times and at a competitive cost per sample. And as food safety standards continue to evolve, laboratories need to be sure that the technology they use today will still meet their needs five years down the line.

Confident Routine Quantitation

Food safety is an evolving field. Technological advances result in ever lower limits of detection and quantitation, and greater insight into the toxicological effects of the chemicals we use in industry and agriculture mean that MRLs are continually being revised. In 2016 for example, the EU announced amendments to regulations governing MRLs for a number of pesticides found in a wide range of products, including the organophosphate insecticide chlorpyrifos.

As food safety standards become increasingly strict, what was once the lower end of a permissible pesticide residue level may be the upper end tomorrow. Laboratories therefore need to be confident that the technology they depend on to quantify these analytes is ready for future challenges.

For instance, high-resolution accurate mass (HRAM) Orbitrap MS from Thermo Fisher Scientific offers sensitivity that can help to safeguard laboratories against changes in MRLs. Hybrid quadrupole Orbitrap mass analyzer instruments combine quadrupole precursor selection with high-resolution accurate mass detection of product ions. The data is acquired at a resolution that can surpass quadrupole-time-of-flight instruments. This selectivity and mass accuracy helps to lower or even eliminate interference and permit lower limits of detection and accurate quantification.

Since MRLs vary for different pesticide-commodity combinations, the techniques used to analyze pesticide residues must be able to identify and quantify analytes over a wide dynamic range. EU limits for pesticide residues in beetroot, for example, vary from 30 milligram/kilogram to as little as 0.03 milligram/kilogram depending on the analyte. The Thermo Scientific Q Exactive Focus hybrid quadrupole-Orbitrap mass spectrometer is an option to meet this challenge, enabling quantitation over a wide dynamic range.

Identifying the Unknown

Routine quantitation of analytes against target lists is incredibly important for the protection of consumer safety, yet for many food safety laboratories it’s only part of the story. As food supply chains become increasingly global and complex, the risk of contamination with previously undetected chemicals becomes greater. However, the detection and identification of these unexpected analytes can be one of the most challenging tasks in pesticide analysis.

While LC-QqQ tandem MS enables highly selective and sensitive quantitation and identification of hundreds of target pesticides in a single run, this approach requires extensive compound-dependent parameter optimization and cannot be easily adapted to screen for untargeted pesticides.

Full scan approaches, on the other hand, are able to screen for a much broader range of analytes, meaning the search is not limited to a pre-defined list of chemical suspects. With the right analytical tools, unexpected analytes can be identified and quantified at the same time as performing routine targeted and quantitative analyses.

However, as full scan approaches produce significantly more data than conventional approaches, it is essential to use data analysis software that can rapidly process results and cross reference against spectral libraries and compound databases in order to make sense of all this information. These software can quickly and automatically search online compound databases such as ChemSpider and mzCloud, or a laboratory’s own database of analytes, to determine empirical formulae or tentatively identify unknown compounds.

Boosting Laboratory Efficiency

With ever increasing numbers of residues to identify, food testing laboratories require robust, reliable, and efficient technologies that enable high productivity. And with budgets a key priority for many lab managers, these analyses must also be performed at a very low cost per sample.

One of the benefits of multi-residue screening based on HRAM Orbitrap MS is the ability to analyze multiple components simultaneously. Combining multiple pesticide workflows in a single run can help laboratories work more efficiently, increasing throughput and boosting productivity. Full scan approaches also allow laboratories to combine pesticide workflows with other types of analyte workflows, such as toxins and veterinary drugs. This way, laboratories can expand the analytical reach of their food testing workflows while minimizing the time and resources spent preparing samples for separate analyses. Furthermore, as HRAM Orbitrap MS approaches also facilitate retrospective analysis, analytes that are not currently on target lists can be identified at a later date without having to store and re-analyze samples.

In addition to advanced hardware, innovative informatics can also streamline workflows and boost productivity. Many forward-looking laboratories are using integrated method development and data analysis solutions that allow operators to conveniently modify pre-configured methods depending on the matrix or analytes of interest. Used in conjunction with cloud-based spectral library and compound database searching, these integrated software solutions can help minimize the time taken between sample injection and the analyst reaching a conclusion.

Simplifying Residue Extraction

One of the most important stages in pesticide quantitation is residue extraction. While full scan analysis workflows are able to screen large numbers of analytes faster and more efficiently than conventional QqQ techniques, they can only do this if the residues they are analyzing are fully extracted from the food matrix in the first place—and if the analytes are chromatographed and ionized.

In recent years, the widespread adoption of extraction techniques such as QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) have simplified the preparation of high-moisture food samples and overcome many of the limitations of conventional approaches. Early approaches typically involved the use of multiple, time-consuming procedures, and produced results that were highly matrix dependent. The QuEChERS method, on the other hand, is based on a single acetonitrile extraction step, with an optional dispersive solid-phase extraction clean-up step. However, although the method is generic, simple to implement, and amenable to a wide range of food samples, the extracts often contain high concentrations of co-extractives.

The latest separation technology is simplifying sample preparation. The Thermo Scientific TurboFlow inline clean-up technique, for example, is a complementary sample preparation approach that eliminates up to two-thirds of the steps required by traditional methods, permitting the injection of complex matrices directly into the instrument. Analytes are separated from the matrix using specialized chromatography columns packed with large particles that retain residues while larger molecules, such as lipids and proteins, pass through. The residues of interest can then be transferred to an analytical column and subsequently analyzed.

As food supply chains become increasingly global and complex, and residue screening workflows require the screening of ever larger numbers of expected and unexpected analytes, food testing laboratories requires robust solutions that can meet not only today’s food safety standards—but tomorrow’s analytes too.

Bromirski is Q Exactive product marketing manager at Thermo Fisher Scientific. Reach him at maciej.bromirski@thermofisher.com.

The post Using HRAM for Pesticide Workflows appeared first on Food Quality & Safety.

When you hear the words “food contamination” your mind makes an immediate connection to unpleasant words such as: illness, disease, unsafe, etc. However, it’s very unlikely that the word “chromatography” comes to mind. One dictionary definition of “contamination” has it as “the action of making something impure by polluting or poisoning.” In other words, the “pure” becomes “impure” by the introduction of something bad that isn’t supposed to be there. Narrowing the definition to the subject of “food contamination,” one definition describes it as “the presence in food of harmful chemicals or microorganisms which can cause consumer illness.” Again something bad has been introduced that shouldn’t be there, which is making the wholesome unwholesome. Food contamination is often divided into two categories: chemical and microbiological. This article will deal only with the chemical contamination of food.*

Chemical Contamination

It is impossible to deal seriously with the subject of the chemical contamination of food without drilling down on some questions, such as the following.

1. What is the potential contaminant?
2. How much is there?
3. Where did it come from?
4. How did it get in the food?
5. What is the specific danger or health risk?
6. How can potential contamination be prevented?

The chemical contamination of food is usually (but not always) quite subtle. Unlike the urgent potato salad incident described in the footnote, the chemical contamination of food is often manifested as trace level exposure to toxic chemicals over long periods of time (i.e., chronic exposure). Potential health effects may not to be realized until many years later, perhaps in the form of carcinogenicity, teratogenicity, and/or metabolic disturbances. And, unlike microbial contamination that can be reversed by such techniques as heating, the chemical contamination of food is generally not reversible. Chemical contamination can only be “cured” by prevention, and prevention is impossible without deep, scientific knowledge about the chemical system associated with the potential for contamination. If you can’t identify, detect, and measure the potential chemical contaminant, you can’t prevent it from happening. You are relying on luck, not science.

Science-Based Prevention

The above concept illustrates why the Food Safety Modernization Act (FSMA) represents such a revolutionary advance in the area of making food safe from chemical contamination. FSMA is wholly anticipatory, not reactionary. You are not allowed to wait decades for a subtle carcinogenic effect to manifest itself before taking action; you must reasonably anticipate the threat of contamination and take proactive measures to prevent it. In other words, you must answer question number 6, mentioned previously. However, you can’t begin to answer this question without reliably answering questions number 1 and 2. For effective prevention, you need to use analytical testing methods that are both qualitatively and quantitatively reliable. The FDA consistently uses the term “scientifically-valid” to describe this basic requirement. Therefore, if prevention is the heart and soul of FSMA then scientifically-valid food testing methods are the means to effective prevention. However, the term “scientifically-valid method” is not a static definition, but a fluid concept.

Food Testing Method Modernization Movement

As technology has advanced, the ability to identify, detect, and measure chemical substances in environmental samples (such as food) has increased exponentially. Arguably, the advance of analytical testing capabilities in the past two decades has exceeded the advance of the prior 100 years. Consequently, food testing methods that may have been the pinnacle of scientific-validity when they were developed 20 years ago may now be quite dated in terms of analytical capability. This is particularly manifested in the inability of many older test methods to adequately differentiate and quantify specific chemical species. This has increased the risk of chemical contamination, particularly in light of the globalization of food supplies that has complicated the tracking of food ingredient origins.

This is probably best illustrated by the unfortunate incident of 2007-2008 where ingredients used in the manufacture of pet food and infant formula were intentionally contaminated (i.e., adulterated) with melamine to fraudulently increase the measured protein content. The scheme initially succeeded because the prescribed test used to measure the protein content (the 100+ year-old Kjeldahl test for total organic nitrogen) can’t distinguish between the nitrogen content of protein and melamine. The Kjeldahl test lacks the ability to speciate specific organic nitrogen compounds and is not fit for the purpose of measuring the protein content of food, at least in the face of a chemical contamination threat from melamine. A sophisticated high performance liquid chromatography (HPLC) test for melamine was subsequently developed, which put an end to that particular contamination threat.

The melamine tragedy brought rapid realization of the vulnerability of many older food testing methods for preventing chemical contamination, whether accidental or intentional. This vulnerability arises from an inherent lack of specificity of older food testing methods—the inability to accurately speciate individual toxic chemical species in a complex food matrix. This inability is particularly stark when one compares the technology underlying the older methods to the much greater capabilities of modern analytical technology. This has led to a broad-based, method modernization effort on the part of government agencies (FDA, NIOSH, EFSA, etc.) and standard setting institutions (AOAC, USP, etc.) to enable the ability to measure, and therefore prevent, the chemical contamination of food. Modern chromatography has played a major role in this food method modernization movement and the ability to prevent food contamination.

Impact of Modern Chromatography

In the introduction to this article, I stated that the term “chromatography” probably isn’t the first thing that comes to mind when considering the subject of food contamination. But, perhaps it should be; at least in the case of chemical contamination. Modern chromatography has an unsurpassed ability to isolate, differentiate, and identify diverse potential contaminants in food. There are many diverse opportunities for food to become chemically contaminated. One needs only to consider the great number of toxic compounds in commerce and the many potential exposure routes from farm to table. The potential for contamination is so diverse, it is impossible to generalize the power of chromatography to prevent food contamination. Instead, I will present a series of thumbnail sketches that illustrate the breadth and depth of recent chromatographic method developments.

The following images are all examples taken from the recently published Phenomenex Food Testing Applications Guide that contains over 150 liquid chromatography (LC), gas chromatography (GC) and solid phase extraction (SPE) applications.

Diversity of Potential Chemical Contamination Scenarios

(click to enlarge)

Mycotoxins: Mycotoxins from cereal based goods by SPE and LC/MS/MS. Produced by certain molds that can grow on grains, mycotoxins are a class of compounds that are highly toxic and carcinogenic.

PAHs: Polycyclic aromatic hydrocarbons (PAHs) in water by GC/MS. PAHs are a class of carcinogenic compounds that arise from the inefficient combustion of petroleum-based products and can contaminate the environment and foods.

PFASs: 23 per-polyfluoronated alkyl substances (PFAS) by UHPLC/MS/MS. PFAS compounds have been widely used in food packaging; they are able to leach into food at trace levels, and since they are extremely bioaccumulative, they can build up in the fat tissue of the consumer.

Melamine: Melamine and cyanuric acid in milk and baby formula products by SPE, LC/MS, and GC/MS. This relates directly to the melamine contamination/adulteration crisis of 2007-8.

Acrylamide: Acrylamide from coffee by SLE and LC/MS/MS. Acrylamide can be found in certain starch-containing foods that have been exposed to heat. Acrylamide is classified as a carcinogen so its presence in food, even at low concentrations, is a concern.

Pesticides in Poultry: Chlorinated pesticides in poultry tissue by SPE & GC/ECD. Chlorinated pesticides are highly persistent in the environment and are also highly bioaccumulative in animal fat.

Pesticides in Spinach: Pesticide residues in spinach by QuEChERS, LC/MS/MS, and GC/MS.

Antibiotics: Antibiotics in meat by LC/MS/MS. Another potential source of food contamination is the introduction of antibiotics and other veterinary products used in livestock production.

Fatty Acids: Fatty acids in powdered infant formula by GC/FID. The analysis of fats in food is generally considered a “nutritional” analysis, rather than a “contamination” analysis. However, with the FDA’s 2016 ban of unhealthy trans fat from processed food, the unlawful presence of trans fat in a processed food such as infant formula would be a case of food “contamination.”

Conclusion

The rapidly evolving science of chromatography has enabled ever more powerful, sophisticated, and effective food testing methods. In turn, these improved methods have greatly strengthened the ability to anticipate and prevent food contamination. Better science-based food testing methods have clearly served to make food safer. And, the science and practice of chromatography is certain to continue its advancement, thereby insuring future improvements in food safety.

Dr. Kennedy, business development manager at Phenomenex, has focused on food safety and environmental monitoring during his over 45-year career. Reach him at Davidk@phenomenex.com.

*Since I am a chemist, and not a microbiologist, I am not qualified to hold a professional opinion on the subject microbial contamination. My only direct experience with the microbiological contamination of food consists of having used my gastrointestinal tract as an indicator of having consumed contaminated potato salad at a family picnic years ago.

The post Using Chromatography to Detect Chemical Contamination appeared first on Food Quality & Safety.

Next time you walk up and down the aisles of your favorite supermarket, think about this—on average 35 percent to 40 percent of all food and fiber crops grown around the world are lost to pests and disease every year. As food safety and risk management professionals, we can all readily appreciate the importance of pesticides in preventing potential food shortages or worse. In fact, pest control dates back to the first person to swat a bug. More methodical methods soon followed. The Sumerians used a sulfur compound to drive off insects. The Egyptians had over 800 recipes for pesticides, while the Chinese used arsenic and mercury compounds to control plant diseases and fend off pests.

The Ubiquity of Pesticides

Though often misunderstood to refer only to insecticides, the term pesticide also applies to herbicides, fungicides, and various other substances used to control pests. Today, more than 5.5 billion pounds of these chemicals are applied to seasonal crops around the world each year. The U.S. agricultural industry alone uses over half a billion pounds of pesticides a year to treat just 21 selected crops, including corn, soybeans, and wheat. According to USDA, about 76 percent of those pesticides are herbicides, 17 percent are soil fumigants, desiccants, and plant growth regulators, while insecticides account for the remaining 7 percent.

With all of those chemicals ending up on global crops, it should come as no surprise to learn that trace amounts of those chemicals end up in the food supply. Remember your mom always telling you to wash that fruit or vegetable before eating it? Turns out she was right. Residual pesticides are found in 52 percent of fruits and over 30 percent of vegetables. But even mom’s advice does not often help, since washing foods does not always remove all of the chemicals. Beyond those that cling to the skin of fruits, vegetables, and grains, some are actually absorbed into the food itself. Despite all of the preventive measures in place, consumers are still eating pesticides on a daily basis.

Even more disturbing is the potential accumulative effects of longtime exposure to these chemicals. The possible implications of exposure to multiple pesticides on food are also of growing concern. It is not uncommon, for instance, to treat crops several times with different pesticides depending upon treatment needs, including insects, rodents, fungi, and soil enhancers. One recent study linked multiple myeloma to certain agricultural exposures, including pesticides, in men throughout North America. Another recent ruling in California will soon require a cancer warning to appear on glyphosate, the world’s most popular weed killing pesticide.

Preventive Measures Abound

In most countries pesticides are highly regulated and designed to dissipate by harvest time, leaving behind only trace amounts of compounds that are measured in the parts per million and billion (ppm and ppb) levels. Government regulators note that those levels are below the legal tolerance limits set by food safety agencies from around the developed world, and are thus safe for human consumption. In every instance, these tolerance levels already factor in an added safety margin that considers their potential impact on children, who consume more food by body weight, as well as people with higher sensitivities.

In order to verify these tolerance standards, farmers, food manufacturers, processors, packagers, and some larger grocery chains now conduct their own testing to make sure every ingredient is within the established tolerance limit. In states like California, which has the strictest standards for pesticide use, testers are mandated by law to fully describe or reference the preparation process and methodologies used as well as provide validation data and all analytical reports upon request.

Testing Methodologies

What do most testing laboratories use to detect, identify, and quantify pesticides in food? While there are multiple methods to measure pesticides at environmentally relevant concentrations, the industry gold standard is chromatography. Both gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) meet the analytical requirements to detect pesticides in food, especially in fruits and vegetables.

GC/MS. This is a highly sensitive and universal detecting system that most people encounter at airports, where it is used to detect substances in luggage or on passengers. Able to detect trace elements down to ppm and ppb, which appear as chromatographic peaks on a chromatogram, GC/MS is frequently used to detect a wide variety of analytes within a single sample matrix, such as pesticide residues in food. GC/MS can also be used to help identify unknown pesticide elements by comparing their relative retention time data to that of a standard, such as chlorpyrifos that is typically used as the standard for common chlorinated hydrocarbon and organophosphate pesticides.

LC/TOF-MS. A newer, more sensitive, and faster technology for pesticide analysis is liquid chromatography/time-of-flight mass spectroscopy, or LC/TOF-MS. Basically, the system determines an ion’s mass-to-charge ratio by measuring the time it takes for an ion to reach a detector that is set at a predetermined distance. That time measures the ion’s velocity and is used to determine its weight, or mass-to-charge ratio, which in turn helps to identify the specific ion. Since LC/TOF-MS collects full spectrum information on samples, the mass spectrometer can examine the data for non-targeted (or unknowns) as well as targeted information that is stored in a spectra database. Using a standard sample preparation procedure like QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe), a LC/TOF mass spectrometer like the PerkinElmer AxION 2 TOF provides lab scientists with the ability to rapidly detect hundreds of commonly regulated pesticides in food at or below the regulatory limit of 10 ppb in concentration. These instruments can also quickly and automatically highlight those residual amounts of pesticides that are above the regulatory limit. LC/TOF technology is an example of how to detect residual amounts of neonicotoid pesticides in honey, which are now the most commonly used insecticide class in the world and are currently under investigation as a possible cause for bee colony collapse disorder.

LC/MS/MS. Liquid chromatography coupled to triple quadrupole mass spectrometry (LC/MS/MS), or triple quadrupole system, is becoming the method of choice for the detection of multiple residual pesticides in food, nutraceuticals, and botanicals. LC/MS/MS systems have a unique detection mode called multiple reaction monitoring, which allows the first quadruple in the system to select the parent ion mass of the analyte before sending them to collision cell for fragmentation. Following this the second quadrupole is able to select daughter ion from those parent ions and send them to the detector for detection. The unique parent/daughter ions combination provides high specificity, selectivity, and sensitivity. Using systems such as the PerkinElmer Altus UPLC system coupled to a QSight 220 triple-quad mass spectrometer can allow lab scientists to identify and simultaneously quantify the trace residue of multiple pesticides in fruit faster than other GC technologies.

In addition, portable GC systems are available when the lab is needed onsite. For example, the 32-pound Torion T-9 GC/MS by PerkinElmer can provide rapid screening of chemicals in food safety applications.

When it comes to flexibility, speed, and accuracy in testing for residual pesticides in food to meet global regulatory requirements there is a wealth of chromatographic options to help make the next family dinner be as pesticide free as possible.

Qin is product manager for food solutions at PerkinElmer. Reach him at feng.qin@perkinelmer.com.

The post Monitoring Pesticides in Our Food appeared first on Food Quality & Safety.

If you’re a laboratory professional who thinks a programmable coffee maker is the greatest thing since sliced bread, you’re in for a pleasant surprise. Not only can you wake up to fresh brewed java at your prescribed time, you can now arrive at your workplace and find freshly made culture media ready and waiting to be used, thanks to the recent development of a new programmable media preparator.

Say hello to the Masterclave 20 Automated Media Preparator, the brainchild of bioMérieux, Inc., Hazelwood, Mo., introduced in November 2016.

“With its automatic water-filling and autostart features, the Masterclave 20 has the ability to prepare fresh agar or broth that is ready when lab operations begin,” says J. Stan Bailey, PhD, director of scientific affairs for bioMérieux Industry.

With this preparator, one medium can be made at a time, during a cycle that takes less than 90 minutes.

“The Masterclave 20 is compact, mobile, and adaptable to any workspace,” Dr. Bailey relates. “A built-in cleaning gun provides fast and efficient cleaning. Our proprietary ‘I Connect’ technology offers fully automated traceability with built-in RFID (radio frequency identification), email alerts, and laboratory information management system (LIMS) connectivity capability. And the instrument is ISO 11133, ISO 7218, and GMP (good manufacturing practices) compliant.”

bioMérieux’s GENE-UP real-time PCR solution for pathogen detection allows for a high level of specificity.

Photo credit: Christian GANET

Another recent bioMérieux offering is GENE-UP, a proprietary real-time three-step polymerase chain reaction (PCR) pathogen detection system, which the company touts is fast, simple, accurate, and requires minimal training.

Dr. Bailey explains that GENE-UP’s first step, sample preparation and enrichment, includes a standardized protocol and workflow with single enrichment and incubation time between 8 and 24 hours. Step two consists of a simplified, generic 5-minute mechanical lysis. (What bioMérieux calls its Magic Cap eliminates the need to cap/decap tubes, so there is less hands-on time required, plus there is a decreased risk of cross-contamination, Dr. Bailey notes.)

The third step, amplification and detection, features the same PCR run for all parameters. “This allows for accurate results within one hour, a higher level of specificity than other molecular methods, and real-time PCR analysis coupled with end-point melt peak analysis,” Dr. Bailey says. “GENE-UP uses a different kind of FRET (fluorescence resonance energy transfer) technology with three levels of specificity, namely primers, FRET probes, and melt peak analysis. This offers an additional level of sensitivity valuable for detecting low level samples.”

Since GENE-UP was introduced in 2016, it has really taken off, Dr. Bailey boasts. “Laboratories are encountering workflow improvements in a molecular platform, equivalent or better data performance than their current method, and with the internal control and melt peak analysis, an immediate value and confidence in the results they are reporting,” he elaborates.

In July 2016, bioMérieux also introduced a new EHEC GENE-UP PCR Kit that combines stx and eae virulence genes, and the top six serogroups in one solution.

Specific protocols (which are simply enriched in prewarmed buffered peptone water) are available for raw milk and raw milk cheese (25 grams), raw beef and veal (25 grams and 50 to 375 grams), and produce (200 grams).

This kit’s EHEC/STEC (enterohemorrhagic Escherichia coli/Shiga toxin-producing E. coli) solution marries GENE-UP in combination with bioMérieux’s long-established VIDAS automated food pathogen detection system to provide what Dr. Bailey describes as “unrivaled specificity.”

“As a result, false positives are dramatically reduced,” Dr. Bailey emphasizes. “That makes this assay a real game-changer.”

BCN Research Laboratories, Inc., Rockford, Tenn., a commercial laboratory that tests food, ingredients, and environmental sponge samples for food manufacturers, has been using GENE-UP since July 2016.

“We typically run about 100 GENE-UP samples every day,” says Amy Pass, BCN’s senior lab technician. “However, one of our clients operates 20 manufacturing plants throughout the U.S. Twice a year, in March and September, that company conducts biannual heavy environmental testing at all of its facilities. So then we are evaluating an additional 150 Salmonella swabs and 150 Listeria swabs per month for each of those 20 plants, which means we are running an average of 400 tests, but up to about 900 to 1,000 tests, per day, during those two months.

“We have a better work flow with GENE-UP compared to other lateral flow methods we have used in the past,” Pass adds. “Since we started using this technology, we have experienced a 25 percent increase in our sample load, but the amount of time our employees spend running the tests has stayed the same.”

Another benefit of GENE-UP, Pass mentions, is that it provides a definite positive or negative result. “So there is no subjective decision looking at the lateral flow strip to see one line or two,” she says.

Colony Tests and Counter

Charm Sciences’ Peel Plate Colony Counter features results in five seconds or less.
Photo credit: Charm Sciences

Results in five seconds or less are one of the charms of Charm Sciences, Inc.’s, Lawrence, Mass., new Peel Plate Colony Counter, which became commercially available in February 2017.

The instrument is designed to analyze a variety of Charm’s Peel Plate microbial tests, which were introduced in August 2015, according to Robert Salter, MS, the firm’s vice president of regulatory affairs.

The tests are prepared media in a shallow dish with an adhesive top. “They are aseptic ready-to-use tests that are simply rehydrated with the food or food dilution, and incubated at times and temperatures appropriate to the microbes being detected,” Salter explains. “Colonies appear as colored spots that are visually counted or automatically counted by the Peel Plate Counter. An air gap between the plate and cover allows colony quantitation, picking and determination of microbial morphology.”

Currently there are Peel Plate tests for aerobic bacteria (Peel Plate AC, introduced in August 2015), coliform bacteria (Peel Plate CC, introduced in 2016), Enterobacteriaceae (Peel Plate EB, introduced in 2017), yeast and mold (Peel Plate YM, introduced in 2016), heterotrophic bacteria in water (Peel Plate HET, introduced in January 2016), and coliforms/E. coli (Peel Plate EC, introduced in August 2015) for use in dairy products, ground meats, other foods, contact surfaces, and water.

“The Peel Plate AC uses standard plate count formulation with 48-hour incubation,” Salter relates. “Peel Plate EC and CC use coliform selective media with enzyme color substrates with 24-hour incubation. The Peel Plate YM (yeast and mold) tests use conventional potato dextrose formulation with three to five-day incubation. The Peel Plate EB (Enterobacteriaceae) tests use selective EB formulation with 24 to 48-hour incubation. And the Peel Plate HET (heterotrophic plate count) test uses R2A formulation for quantifying bacteria in water with three to five days incubation.”

The new Colony Counter reads all of the aforementioned Peel Plates, Salter says.
Additionally, Charm offers a high volume version of Peel Plates CC-HVS, YM-HVS, EB-HVS, and EC-HVS, designed for a 5 milliliter (ml) sample, typically used for food plant sponge sampling of the production environment, for greater sensitivity in ready to eat foods, and to test water. “These are viewed visually and not yet supported by the Peel Plate Counter,” Salter says.

Salter believes the Peel Plate offerings are an improvement on many existing simple-to-use microbial test products. “The Peel Plate provides a ready-to-use platform that is self-wicking, stackable, and resistant to sample pH/disinfectant effects,” he relates. “Charm Peel Plate tests are simple to interpret color spots that are specific without the need to confirm, but will allow traditional picking of colonies for additional microbial isolation, testing, and identification.

“Peel Plate CC and EC tests use traditional gram negative selective media with bacterial species specific color producing enzyme substrates and a 24-hour incubation,” Salter elaborates. “Coliforms produce easy to interpret red colonies, and E. coli blue colonies, that do not need additional confirmatory steps or difficult to interpret and time dependent gas production.”

The Peel Plate EC test holds Performance Test Method (PTM) Status 061501 with the AOAC Research Institute for total coliform in dairy products tested at 32 degrees Celsius and for E. coli and coliform detection in water, surface rinses, environmental sponges, and foods such as ground meats, eggs, chocolate, and dry dog food tested at 35 degrees Celsius.

Peel Plate AC uses a red color indicator for aerobic bacteria growth in a 48-hour incubation. It holds PTM certificate 071501 for dairy products at 32 degrees Celsius and ground meats, eggs, chocolate, environmental sponges, and dry dog food tested at 35 degrees Celsius.

Peel Plate YM uses a blue/green indicator for yeast and mold growth in a 3- to 5-day incubation. It holds PTM certificate 061601 for fruit, juices, dairy, flour, tortillas, hummus, and environmental sponges.

Based on additional multi-laboratory reference method comparative data, the Peel Plate EC test and the Peel Plate AC test were voted in the 2015 National Conference on Interstate Milk Shipments for inclusion into the Pasteurized Milk Ordinance governing U.S. milk testing requirements.

“Many of our customers are using Peel Plate tests to verify their sanitation and hygiene practices and to monitor and improve food product shelf life,” Salter notes. “They are competitively priced with other simple and ready to use microbial methods, saving time, skill, and labor required by the traditional microbial methods.”

The Colony Counter is a plug and play computer/camera/software that provides complete data management with a real-time print option, Salter points out. Date, time, operator, sample ID, test type, test matrix, count, sample dilution, calculated colonies/ml or gram product, raw plate image, and processed plate image are stored in folders and hyperlinked in a SQL database spreadsheet. Barcode scanning capability provides for a simple one button analysis.

“Onboard Ethernet enables real-time downloading to network SQL databases,” Salter says. “The images are saved as .jpeg files and are viewable at a later date. All data is reviewable by day, week, and month with the touch of a button. It provides for a simple integration with LIMS.

(Without the Peel Plate Counter, Peel Plate tests are scored based on visual counts of colonies, Salter notes. “Visual count is how the methods were compared to the reference methods—also visual—for the approvals,” he says.)

“Inasmuch as food companies are updating their microbial sanitation verification and their end product microbial monitoring programs to address the new Food Safety Modernization Act regulations, we believe the Peel Plate Counter is a valuable tool to assist in automated documentation and record keeping that will enable food production stakeholders to more easily meet audit and inspection requirements,” Salter adds.

Molecular Detection Chemistries

Roka Bioscience’s fully automated Atlas System enhances the accuracy, speed, and efficiency of testing through detection of molecular pathogens.

Photo credit: Roka Bioscience

Roka Bioscience, Inc., Warren, N.J., stands out in many state-of-the-art food laboratories by offering differentiated molecular chemistries for pathogen detection. One such cutting-edge chemistry is target capture, Roka’s proprietary sample preparation method that is integrated into the company’s fully automated testing instrument called the Atlas System.

Simply stated, target capture uses highly specific nucleic acid hybridization to purify and concentrate only the target RNA of interest, according to W. Evan Chaney, PhD, Roka’s director of customer applications and microbiology.

“Roka’s target capture technology is the only fully integrated nucleic acid based sample preparation technology in the industry,” Dr. Chaney says. “This technology is the initial step in all of Roka’s automated assays and serves to not only add specificity, but to also clean up the sample prior to detection.”

The diversity of sample matrices in food related analyses results in very unique diagnostic application challenges, Dr. Chaney points out. “Our target capture technology helps to address these challenges by providing an ideal sample for downstream amplification and detection by molecular chemistries called transcription-mediated amplification (TMA) and hybridization protection assay (HPA),” he relates.

An RNA based amplification system, Roka’s TMA has been used in clinical diagnostics for many years and was first commercially introduced to the food industry in 2012.

“TMA is still novel within the food industry and many food safety professionals are not aware of the differences between it and incumbent testing methods, like PCR,” Dr. Chaney says.

TMA uses two enzymes to drive the reaction, RNA polymerase and reverse transcriptase, he explains. According to Dr. Chaney, TMA is very rapid, resulting in a billion-fold amplification of the target RNA within 15 to 30 minutes.

“One component of this efficiency is the greater abundance of RNA target in cells as compared to DNA,” he says. “TMA is different from older DNA based chemistries such as PCR in that it is isothermal and autocatalytic. The higher RNA copy number per cell, combined with TMA, results in a very robust amplification that may occur in a shorter timeframe as compared to PCR, which can translate into quicker turnaround times for results.”

All rapid methods have various analytical limits of detection in the enriched sample, Dr. Chaney points out.

“For example, most PCR methods require 10⁴ or more cells per ml, whereas, TMA only requires 10² to 10³,” he says. “This becomes quite important to prevent false negatives when considering industry’s move to larger sample sizes and reduced incubation times across an increasingly diverse and complex range of matrices.”

Post TMA, all Roka assays detect any amplified product utilizing HPA, which Dr. Chaney describes as a highly specific chemiluminescent reaction from which the intensity is measured by the Atlas instrument.

“In addition to the detection of any pathogen, each individual sample processed by Roka’s technology includes an internal amplification control, which ensures all reactions occurred,” he says, adding that all of these technologies are automated on the Atlas instrument.

“Our chemistries and controls, coupled with integrated sample preparation on a fully automated platform, translate into faster result times, laboratory efficiency, full traceability, and more accurate foodborne pathogen screening results, particularly for challenging sample matrices,” Dr. Chaney elaborates. “Roka’s technology is routinely utilized in many industry segments, including commercial laboratories, poultry, ready-to-eat meats, produce, dairy, confectionary, ingredients, cereals, multi-component foods, snack foods, and as a tool in pre-harvest food safety.”

TMA is routinely used by Marshfield Food Safety, LLC (MFS), Marshfield, Wis., a firm that specializes in providing customized, onsite process control laboratory services for U.S. food processing operations.

Holding accreditation to ISO 17025:2005 standards with the American Association for Laboratory Accreditation at all nine of its U.S. food testing laboratories, the MFS portfolio includes an extensive list of microbiology and chemistry laboratory test offerings.

“We have been using TMA for qualitative, semi-quantitative, and limits testing for Salmonella for four years,” says Roy Radcliff, PhD, chief executive officer, MFS. “We started using TMA for identifying Listeria species in early 2016, and we have been using it for L. monocytogenes since August 2016.”

Dr. Radcliff says that the ease of use and decreased hands on time are benefits of TMA enjoyed in MFS labs.

“Roka’s Atlas System integrates well with our LIMS,” Dr. Radcliff relates. “The automatic importation of results into the LIMS and tracking of TMA kit lot numbers simplifies the workflow and traceability, which in turn makes them easily auditable.”

Dr. Chaney mentions that, along with continually expanding its footprint by developing unique applications for its current food testing products, Roka endeavors to provide support to its customers, and also works with strategic partners. “We strive to partner with the industry we serve in collectively advancing food safety,” he emphasizes.

Roka’s menu of automated pathogen detection kits includes Listeria spp., L. monocytogenes, Salmonella, Escherichia coli O157:H7, and non-O157 Shiga toxin-producing E. coli, as well as applications including semi-quantitative Salmonella. All of these kits utilize target capture, TMA, and HPA molecular chemistries, and these kits are utilized in Roka’s new applications, Dr. Chaney says.

“More recently, we have introduced a new kit for detection of Listeria spp. specifically in environmental samples, in addition to a novel workflow for use in mitigating the diagnostic challenges that may arise with use of industrial phage based processing aids,” Dr. Chaney adds. “We are currently validating some new and exciting options, such as media alternatives and new assay application parameters for our customers that we anticipate will confer a number of benefits and efficiencies to their operations.”

MALDI-TOF

As general manager of Mérieux NutriSciences, Wendy McMahon, MS, CFS, oversees the company’s Silliker Food Science Center (SFSC) contract research laboratory, Crete, Ill.

McMahon believes matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectroscopy (MS) is an important tool for bacterial and fungal identification in food laboratories today. “It’s really used for determining unknown organisms, mostly spoilage and contaminations, with mold being a good example,” she points out.

Available commercially for less than 10 years, MALDI is a three-step soft ionization technique that allows the analysis of biopolymers such as DNA, proteins, peptides, and sugars, and also large organic molecules. The TOF is the type of mass spectrometer most widely used with MALDI, primarily because of its large mass range.

McMahon says it’s interesting that the microbiology world is using MS for bacterial identification, since MS is a tool used for chemical analysis. “Chemists get a kick out of this,” she quips.

Under McMahon’s leadership, the SFSC is launching the use of MALDI-TOF in the lab during the spring of 2017. “We expect hundreds of ID requests per month due to its quick time to result,” she predicts.

The SFSC is using bioMérieux’s VITEK MS to run its MALDI-TOF tests. “We made that decision based on the database,” McMahon relates. “Specifically, bioMérieux’s database has been established with an average of greater than 14 isolates per species and an average of 26 spectra per species, making it very specific. If an organism is not a part of the database (unidentifiable), then 16S ribosomal RNA (rRNA) gene sequencing can be used for identification.”

The time to result was also a deciding factor in selecting VITEK MS, McMahon adds, noting that it allows for faster investigations and decisions than getting identifications with gene sequencing affords.

“Microbiologists appreciate the quick turnaround time MALDI-TOF offers, less than 30 minutes once the isolate is ready, while requiring very little hands on time from a technician,” McMahon elaborates. “In contrast, the gold standard of 16S rRNA gene sequencing for bacterial identification takes a day of operations and a significant amount of hands on time.”

The SFSC has been using the 16S rRNA method for more than 10 years.

“MALDI-TOF is becoming more widely used throughout the food industry due to the quick results and ease of use,” McMahon says. “The clinical and pharmaceutical industries took to it first and the food industry is quickly catching on. MALDI-TOF’s use in food will increasingly provide companies with faster results when investigating spoiled product, mold contaminations or out of specification raw ingredient or finished product.”

Details to Work Out

There are details to work out in the increasingly more sophisticated world of food laboratory technology, especially with regard to the pathogen testing and detection end of things, says Lee-Ann Jaykus, PhD, the William Neal Reynolds Distinguished Professor in the Department of Food, Bioprocessing, and Nutrition Sciences at North Carolina State University, Raleigh, and also the scientific director of the USDA-NIFA Food Virology Collaborative.

“In recent years, several assays have been designed to meet the need of providing testing results in near real-time (same day), but by and large, they still require some cultural enrichment for pathogen detection, even though enrichment may be abbreviated,” Dr. Jaykus relates.

To get true real-time (in a matter of minutes) pathogen detection will require methods that are completely culture-independent, she says.

“Such pathogen detection will also require pre-analytical sample processing methods, also called ‘sample prep,’ to concentrate the organisms from the sample matrix, and remove matrix-associated inhibitory compounds,” she elaborates. “While some novel sample prep technologies have been launched in the past several years, no silver bullet has been found yet.”

Many groups, be they academic, industry, or government, are actively developing biosensor technologies, Dr. Jaykus points out. “Many of these technologies are novel and ‘sexy’ but still do not have the low detection limits necessary for pathogen detection in foods,” she says. “In addition, the sample matrix can be a significant impediment to analytical sensitivity. Another reason for sample prep, and a personal caution, is that without one (sample prep) we cannot have success in the other (biosensors).”

Dr. Jaykus believes that as detection become less dependent upon culture and more dependent upon nucleic acid sequence, the issue of bacterial cell or virus viability becomes more important.  “Just because we can detect DNA does not mean that the organism is alive,” she notes. “This issue is of importance in making decisions about prevention and control in food safety, as well as management of recalls and outbreaks. It has not yet been resolved.”

Metagenomics

Metagenomics, a term that reportedly first appeared in peer reviewed literature in 1998 (Handelsman et al), basically the study of genetic material recovered directly from environmental samples, promises to impact laboratory analysis with ever increasing significance. In 2005 Chen and Pactor defined metagenomics as “the application of modern genomics technique without the need for isolation and lab cultivation of individual species.”

What some scientists call the metagenomic revolution has resulted in a lot of DNA sequence data for various foodborne pathogens, Dr. Jaykus says, while emphasizing that, “relative to the volume of data available, we currently do not have the critical mass of scientists necessary to interpret it. We are also not entirely certain as to the practical use of those data in food safety management. This will become clearer in years to come, but the field is currently in its infancy stage.”

The post Food Laboratory Breakthroughs appeared first on Food Quality & Safety.