Food safety is a growing global concern; in fact the food safety testing industry is estimated to reach $15 billion by 2019. While a large portion of this market involves pathogen testing, contaminant testing by techniques such as gas chromatography, high-performance liquid chromatography, and liquid chromatography coupled to tandem mass spectrometry is a growing sector because these techniques provide rapid results and allow analysts to detect and quantify contaminants at extremely low concentrations. While the actual chromatographic analysis is a rapid procedure, samples must be properly pretreated prior to analysis, which can add significant time requirements. As concerns over food safety grow, laboratories must process their samples as quickly as possible, making it extremely important to determine the quickest and most effective sample preparation technique.

(left) Image 1: Initial sample prior to cleanup by SLE. At left, coffee with 2% ammonium hydroxide. At right, control ground coffee.
(right) Image 2: Sample after cleanup by SLE. At left, clean sample after extraction. At right, Novum SLE 6 cc tube after extraction.
Image Credit: Phenomenex

There are many sample preparation techniques available to food safety analysts, each of which has its pros and cons. The most effective sample preparation technique is perhaps solid phase extraction (SPE) because it results in extremely clean, concentrated samples; however this technique requires a significant amount of method development time. A more popular technique in the industry is to perform a liquid-liquid extraction (LLE). LLE is fairly quick, does not require as much method development time as SPE, and produces a rather clean sample. To perform LLE, the food sample is first homogenized into a liquid form. Once liquid, the food sample is mixed with a water-immiscible organic solvent by shaking the two solvents in a flask or separatory funnel. During the shaking process, target analytes partition out of the aqueous sample and into the water-immiscible solvent, leaving behind interferences such as lipids, proteins, and salts. The water-immiscible solvent can then be collected by manually separating this layer from the aqueous layer. While this procedure is rather easy to perform, it does introduce challenges such as analyte loss due to emulsions, or bubbles, that can form at the interface of the two liquid phases as well as incomplete collection of the water-immiscible layer during the liquid separation process. These challenges also make the technique difficult to automate, which can eliminate the ability to perform high-throughput sample processing.

This technique ­eliminates the formation of ­emulsions and the need to manually ­separate liquid phases.

Supported liquid extraction (SLE) has become a popular means to avoid the challenges associated with LLE in bioanalytical laboratories; however this technique has not yet been rapidly adopted in the food safety industry. The technique traditionally relies on diatomaceous earth to provide a solid support on which a liquid separation can be performed. Aqueous-based sample is loaded onto the sorbent, which acts like a sponge to distribute the sample across the surface and inside the pores of the sorbent. Water-immiscible solvent is then added to the sorbent and the two liquid phases interact, allowing target analytes to partition into the water-immiscible solvent, which then drips out of the sorbent and into a collection vessel. This technique eliminates the formation of emulsions and the need to manually separate liquid phases. In addition to the ease of use over LLE, SLE can also be automated, which provides further timesavings, particularly for high-throughput laboratories. Traditional SLE products are packed with diatomaceous earth which is a natural product made up of fossilized diatoms. The material can be found in many different mines across the world, and variances in the product can occur if it is mined from multiple locations. To eliminate the potential challenges such as consistency and performance variances, a synthetic SLE sorbent has recently been engineered by

(click for larger image) Table 1: Recovery and % CV of acrylamide.

Phenomenex—Novum SLE. With a synthetic SLE sorbent, scientists can expect consistent results from batch to batch due to the stringent manufacturing and QC processes behind the lab-engineered product. This consistency and reliability is important when analyzing low-level contaminants such as hazardous compounds in food.

To test the effectiveness of the new, synthetic SLE sorbent in a food safety testing setting, acrylamide was extracted from both ground coffee and instant coffee.

SLE Extraction Protocol

1. Coffee pretreatment: Ground coffee, 40 milligrams (mg)/milliliter (mL)
   • 60 grams of ground coffee was percolated with 1,500 mL of boiling water Instant coffee, 8 mg/mL
   • 2 grams of instant coffee was dissolved in 250 mL of boiling water Coffee was allowed to reach room temperature and was then spiked with acrylamide to reach 100 nanogram (ng)/mL (ground coffee) and 200 ng/mL (instant coffee) by adding 20 microliter (µL) acrylamide-13C3 (4 microgram/mL in water) to 800 µL of the prepared coffee. The 150 µL of 2 percent ammonium hydroxide in water was added to the spiked samples that were then vortexed for 30 seconds.
2. Load the pretreated sample onto a 6cc SLE tube.
3. Apply 5 inch Hg vacuum for 5 to 10 seconds to initiate flow into the sorbent.
4. Wait 5 minutes.
5. Load 2x 2.5 mL ethyl acetate/tetrahydrofuran (1:1) and collect under gravity in a collection tube that contains 10 µL ethylene glycol.
6. Apply 5 inch Hg vacuum for 20 to 30 seconds to complete elution.
7. Dry down and reconstitute in water.

After extraction there was an immediate visual difference between the initial sample (see Image 1) and the clean sample (see Image 2). The resulting clean sample was then analyzed by LC/MS/MS, which resulted in acrylamide recoveries of 94.9 percent from the ground coffee and 92.8 percent from the instant coffee, indicating that the SLE cleanup was not only effective at removing interferences but was also resulted in excellent analyte recoveries. In addition to high recoveries, the method produced % CV values of 0.78 (ground coffee) and 1.61 (instant coffee), which suggests that the extraction is also reproducible (see Table 1).

As the food safety industry continues to grow and monitor an increasing list of contaminants, testing laboratories must find effective and reliable ways to analyze a variety of food samples. Sample preparation is crucial when working with food samples because of the many complex components within the sample; however this is perhaps the most time-consuming step of the analytical process. The introduction of SLE to food safety testing is providing labs with a rapid yet clean solution to sample preparation that is not only consistent and reliable but can also be automated for further timesavings.

Pike is the strategic marketing manager of new products at Phenomenex. She received her Bachelors degree in biochemistry and molecular biology and her Masters degree in biotechnology from Boston University, Mass. Reach her at EricaP@phenomenex.com.

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Waters Corp. is targeting food safety and other applications with its new system that bypasses traditional timely sample preparation using a hollow blade similar to a surgeon’s scalpel coupled with software and a mass spectrometer.

Called the Rapid Evaporative Ionization Mass Spectrometry (REIMS) Research System with iKnife Sampling, it can help researchers quickly differentiate samples and identify their features, according to the company, giving them more insight into the chemical and biological systems they are studying.

Its biggest differentiator from traditional liquid chromatography mass spectrometer (LCMS) and molecular techniques like polymerase chain reaction is that is works without requiring sample preparation. The iKnife cuts a heated sample, forming a vapor rich in chemical information. The iKnife is about 1 millimeter thick and 2 centimeters long. The vapor moves through it and an attached 3 meter long tube to a transfer capillary, where molecules are ionized at a heated impactor surface and potential contaminants are removed. The ions are analyzed by time-of-flight mass spectrometry (TOF MS) to get a molecular profile.

“LCMS depends on taking a sample. If you take a sample from a fish, for example, you must homogenize it, centrifuge it and filter it. It’s quite a manually intensive process, and a lab technician has to be sitting with it for some hours,” says Mike Wilson, PhD, who is product manager of benchtop TOF MS at Waters’ office in Manchester, U.K.

Waters acquired the REIMS technology from MediMass Ltd., of Budapest, Hungary, in July 2014. The company was in a three-year collaboration with MediMass and Imperial College London focused on advancing the technology.

Dr. Wilson says the system will be available as both a kit listing for about $40,000 that can be added to installed Waters Xevo and SYNAPT mass spectrometers. The company also will sell systems including the iKnife and a box generator, an ion source mass spectrometer and Progenesis QI software starting at around $490,000. The total system cost depends on the technology in the package.

Dr. Wilson says the product could be used both to glean information about a species of meat or fish and potentially to detect food adulteration. He points to a 2013 scandal in the U.K. when horsemeat was found to be mixed in with beef. The REIMS with iKnife technology could be used to test first pure beef and pure horsemeat, and then to understand the mixed sample quickly.

The company claims the analytical tool may help the food safety industry come closer to a real-time result.

Dr. Wilson expects initial customers to be researchers in universities and institutions focused on food analysis.

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Consumers have never been more aware of food safety issues. A quick Google News search for “food safety” turns up headlines from around the world. In addition to the usual suspects such as botulism, E. coli, and Salmonella, consumers worry about pesticides and other chemical contaminants in their foods. Another fear is food bioterrorism.

Just the mere suspicion of a contaminated product has far-reaching consequences for a food supplier. Because testing food samples (whether it is to look for contaminants or to examine nutritional components) can help find irregularities before products reach consumers, it is a vital activity in the food industry.

According to Mark Carter, general manager of Silliker, Inc.’s Food Science Center (Homewood, Ill.), sample preparation is the most overlooked aspect of food safety. “A lot of work gets done on the back end in detection or enumeration, but we tend to neglect the sample prep area,” he says. Sample prep needs to be validated and optimized along with test procedures and should be re-examined every time any change is made to verify that the procedures being used are still applicable.

“There are a lot of directions to consider,” Carter says. “For example, how do you categorize your product? Is it meat, poultry, dry powder, or liquid? How you begin obviously depends on what you’re testing and what you’re testing it for. Currently, there is no compendium.” Some people, including Carter, would like to change that.

Desperately Seeking a Compendium

A working group of 19 met in July at the International Association for Food Protection’s (IAFP) Annual Meeting, held in Lake Buena Vista, Fla., to discuss the state of sample prep in the food industry. Led by co-chairs Mary Lou Tortorello, PhD, of the U.S. Food and Drug Administration (FDA), and Lee-Ann Jaykus, PhD, associate professor, North Carolina State University Food Science Department, the group’s long-term goal is to create a compendium for sample test methodology in the food industry.

“This meeting was the kick-off to decide what we will try to do,” says Tortorello. “A compendium is a very long-term goal. We discussed what considerations need to be made if we want to improve sample prep to the point of linking it up and integrating it with detection or ID assays in an integrated, automated system. Initially, we’ll focus on microbiologic testing; we won’t be looking at toxins.”

Tortorello points to the many inconsistencies that can occur, depending on which agency is involved. “Whether you are talking with the U.S. Department of Agriculture [USDA], FDA, Health Canada, or some other agency, you might find very different ideologies and methodologies in sample prep. The first thing the group would like to see is consistency, and the second is to move into that integrated, automated mode within each commodity area.”

Commodity Areas Defined

Although there were differences within the group, it has been able to define commodity areas based on guidelines from the Association of Analytical Communities. The group’s commodity list includes meats; poultry; dairy; eggs; fruits, vegetables, and nuts; seafood; cereals, grains, and pasta; bakery, chocolate, and confectionery; and dressings, condiments, and spices.

“We decided to assemble a team of experts for each commodity,” Tortorello says. “These experts will examine the status of sample prep in their area and decide how to make it more consistent. Each expert group will recommend the best methods for achieving the goal of an integrated, automated approach to sample preparation.”

The working group’s first output will be a white paper authored by five of the 19 members of the working group. Meanwhile, other members of the group are focusing on recruitment. The working group participants are all volunteers who expressed interest in the area of sample prep.

Tortorello and Jaykus started by e-mailing the list of participants in the applied methods segment of the IAFP Annual Meeting. Nineteen of them responded favorably. The group consists of members from industry, regulatory agencies, and academia and hopes to attract more interest with the white paper.

Tapping Universities

While members of the food industry would have an obvious interest in the topic, Tortorello points out that there is a great deal of expertise available in academia as well. She hopes to draw on the knowledge of both groups. Tortorello adds that she is not working with the group in an official FDA capacity and that the FDA is not overseeing the working group. She is, instead, a member of the working group representing her own interest in the issue.

During the initial meeting, the working group debated how to choose sample prep methods and collect samples, discussed the current information gaps, and tried to define needed research and improvements. “We didn’t always agree on things,” Tortorello says. “It was a lively discussion.”

Also meeting for the first time was a subcommittee of the National Advisory Committee on Microbiological Criteria for Foods (NACMC). It is charged with determining the most appropriate technologies for the USDA’s Food Safety and Inspection Service (FSIS) to adopt in performing routine and baseline microbiological analyses. The group will develop guidance and recommendations for the FSIS for improving both laboratory and in-plant testing methods for pathogens and indicator organisms.

According to an NACMC update, “this project will assist the agency with its goal of moving into the next generation of microbiological testing methods and will focus on exploring the utility of new technologies.”

The formation of the working group to put together a compendium for sample testing follows the FDA’s July 2007 launch of the Manufactured Food Regulatory Program Standards, which were designed to encourage state agencies to adopt more uniform enforcement of FDA regulations. The program is voluntary, but the agency hopes it will lead to more consistent enforcement across the United States.

That enforcement is crucial; foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States each year. These program standards are expected to set out best practices for protecting consumers from such foodborne illnesses.

McLeod is a freelance writer based in Eugene, Ore.

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