(Editor’s Note: This is an online-only article attributed to the December/January 2019 issue.)

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Food contamination is a serious problem on a global scale. Monitoring the presence of persistent organic pollutants, such as dioxins, is crucial to reduce risk to human health and maximize food safety. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are a common contaminant and have been shown to exert a number of toxic responses, causing reproductive and developmental problems, immune system damage, and hormone interference, and can be carcinogenic. Detection and quantification of dioxins in feed and food supplies is of vital importance, as more than 90 percent of human exposure to dioxins is through food, in particular meat and dairy products, and fish and shellfish.

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The average home cook chops an onion just before using it, but those who cut onions in advance—whether for home or industrial cooking—have long noticed a peculiar occurrence: Waiting a while to use onions after cutting them often results in their developing a bitter taste that was not previously present when they were freshly cut.

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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

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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.

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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.

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Metrohm expands Ion Chromatography instruments and accessories supported by Waters’ Empower Chromatography Data Software. With the release of new drivers the company adds support for more sample handling accessories, IC systems with amperometric and conductivity detection, and dual-channel systems for combined anion and cation analysis. Metrohm integrates with Empower to control all aspects of the system, from a small volume autosampler to detection schemes. Users have the ability to determine anions, cations, and polar substances by ion chromatography and suppressed conductivity detection in concentrations ranging from percent to ultratrace. Metrohm USA, Inc., 866-638-7646, www.metrohm.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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Monitoring pesticide residues in fruit and vegetables remains a key priority for international food safety. Increasing imports from countries, such as China and India, with substantially different regulations to their Western counterparts, highlights the need for stringent pesticide monitoring. Tandem mass spectrometry coupled to chromatography systems, such as gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS), operating in multiple reaction monitoring (MRM) mode has emerged as the industry standard for monitoring residues in fruits and vegetables. However, a continuing challenge in multi-residue analysis is finding a sample preparation method that is as easy, fast, and cost-efficient as possible.

The U.S. FDA recommends the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method for residue screening based on MRM with advanced gas or liquid chromatography coupled mass spectrometry systems. However, these QC methodologies can be complex and time consuming, particularly for trace pesticide analysis in complex biological matrices. A modified QuEChERS preparation protocol developed by the FDA Irvine laboratory in California can extract multiple classes of pesticides from a wide variety of samples. This methodology presents an alternative to the conventional QuEChERS technique and allows the extracted matrix to be directly injected into the instrument, saving preparation time. This article demonstrates that the modified QuEChERS sample preparation protocol is a simple, less expensive, and unified alternative to conventional QuEChERS protocol.

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Figure 1: A modified QuEChERS sample preparation protocol developed at the U.S. FDA Lab at Irvine.

Challenges of International Trade

The liberalization of global trade has greatly benefited emerging economies around the world. Following its accession to the World Trade Organization in 2001, China is now a major global producer of agricultural products, especially fruits, vegetables, rice, and pork. In 2009, China was the fourth leading global agricultural exporting country (behind the U.S., Brazil, and Canada), with exports to the U.S. alone reaching approximately $3.3 billion in 2010. However, the lack of global standardization or global consensus on the use of pesticides is a barrier that limits producers from accessing the full potential of the export market. Imports from regions where pesticide use is less restricted to those with stringent regulations are frequently subject to detention and often returned to their country of origin or disposed of, resulting in an immediate loss in investment.

The benefits for global standardization have now been recognized and governments around the world are beginning to take steps to bring the regulations guiding crop growth and maintenance in line with their Western counterparts. For instance, China has recently limited the use of harmful pesticides that are widely banned on international markets, while Pakistan has announced its intention to bring rice and mango production in line with FDA guidance. However, accurate and robust pesticide quantification methods are essential to determine whether consumable products comply with international and domestic regulatory standards.

Extracting multiple pesticide species from fruits and vegetables is a challenging process due to the complex biological matrices. Conventional analytical techniques have, until recently, been unable to deliver the sensitivity required to achieve reliable trace level analysis. Consistently achieving the high levels of sensitivity required can also be a time intensive and laborious task, which is undesirable for a high-throughput routine QC laboratory. Developing analytical technologies and screening methodologies to be able to quickly and accurately qualify and quantify trace pesticide residues is therefore a priority for both instrument developers and regulatory bodies.

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Figure 2a (left): MRM chromatograms for selected pesticides at 1 ppb in spinach extract by LC-MS/MS; Figure 2b (right): MRM chromatograms for selected pesticides at 5 ppb in spinach extract by GC-MS/MS.

Advancing Detection Procedures

The USDA QuEChERS method for pesticide residue analysis was presented at the 2002 European Pesticide Residue Workshop. QuEChERS simplifies the analysis of pesticide residues in food products, including fruits and vegetables, and takes advantage of advances in LC-MS and GC-MS. The method is now standardized within AOAC 2007.01 EN15662, having proven more productive than conventional preparation techniques.

The QuEChERS protocol uses less expensive and fewer solvents and provides a faster extraction method. Samples are homogenized via blending before centrifugation and extraction with a suitable reagent. The modified QuEChERS methodology developed by the U.S. FDA laboratory in Irvine presents an even simpler alternative to the conventional QuEChERS technique, allowing the extracted matrix to be diluted and injected directly into the GC/LC-MS to save further time.

Advances in both GC-MS and LC-MS have led the U.S. FDA to recommend them as the platform for QuEChERS screening. Modern LC-MS systems are generally considered to be more powerful and are able to separate a greater range of pesticide products. However, where routine detection of known volatile analytes is required, GC-MS systems are a suitable and lower priced alternative.

Despite the increased availability of sophisticated GC-MS and LC-MS systems, the functionality of many commercially available systems is still limited by technological aspects, struggling to deliver the levels of sensitivity and specificity required. Yet, triple quadrupole MRM overcomes many of these limitations and delivers the performance levels required for pesticide detection.

Technological Developments in GC-MS and LC-MS

MRM helps to maximize reliability in pesticide detection by fragmenting ionized analytes into multiple ions. When MRM is incorporated into GC-MS and LC-MS, there is a dramatic increase in signal to noise ratio, greater specificity, and better quantitative performance. Hardware advances in both GC-MS/MS and LC-MS/MS have refined the performance of triple quadrupole MS for pesticide detection to ensure high performance is maintained throughout high throughput analysis. In GC-MS/MS, an axial ion source reduces the contact of ions with hot surfaces and avoids the matrix build-up on the ion source. Higher signal to noise ratio is maintained, reducing the need for instrument cleaning and the resulting downtime, while ensuring high performance is maintained, crucial for a high-throughput laboratory.

Design advances in LC-MS/MS also deliver similar improvements in robustness and sensitivity by optimizing ion transfer. The systematic loss of sensitivity resulting from residue deposition is overcome by use of an open orifice rather than a capillary interface between the liquid chromatography and mass spectrometry elements. An Active Exhaust further reduces chemical noise and increases sensitivity and specificity of trace analyte detection by reducing gas recirculation within the ion source.

A further point of development has been extending LC-MS/MS to cover thermally labile pesticide species that commonly breakdown during liquid chromatography eluent “over-heating” prior to nebulization. This is achieved by the incorporation of a vacuum insulated probe within the ionization unit and around the liquid chromatography eluent to reduce heat transfer to the sample. Vacuum Insulated Probe Heated Electrospray (VIP-HESI) technology ensures high signal-to-noise ratios, superior robustness, and broadens the analysis range of liquid chromatography techniques.

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Table 1: MRM transitions of 30 pesticides by GC-MS/MS and LC-MS/MS systems.

Advantages of CBS

Developing a multiple reaction monitoring method can be a time consuming task as there can be hundreds of pesticides to identify in a single run. Traditionally a chromatographic run is divided into fixed segments and only the MRMs eluted in each segment are monitored. However, residues eluting near the joint of the two adjacent segments, MRMs must be set up in both segments to assure detection. The need for duplicated MRMs leads to slower duty cycles that must be carefully optimized to ensure sensitivity is not lost from short dwell times. Compound Based Scanning (CBS) streamlines method development for multi-residue analysis. Following a number of initial runs to locate the retention time window for each compound, the optimal scan time is automatically calculated by the software, which processes all the overlapped retention time windows. In this way, the duty cycle is optimized and fixed segments are no longer required, which is greatly advantageous in a high-throughput laboratory.

The CBS workflow focuses on the compounds rather than the individual MRMs. The MRM transition for an analyte does not need to be known. Instead the software auto-fills this information from a compound library containing more than 2,500 MRM transitions covering more than 900 contaminants. Each compound library is then linked to retention time, primary and secondary MRM transitions and collision energy. Using this library, CBS dramatically reduces the time taken to set the initial MRM methods to give accurate data while simultaneously improving workflow productivity.

The below case study explores how LC-MS/MS and GC-MS/MS systems incorporate these hardware and software developments to provide robust, fast, and simple analysis of complex food matrices.

Rapid Pesticide Analysis using LC-MS/MS and GC-MS/MS

Three vegetable matrix samples of rice, avocado, and spinach, representing low moisture content, fatty content, and high moisture content vegetable groups respectively, were extracted using the modified QuEChERS protocol developed at the U.S. FDA Lab at Irvine, shown in Figure 1.

Thirty pesticides amenable for both GC-MS and LC-MS were spiked into three extracted vegetable matrices. Calibration solutions were diluted using extracted blank matrices and prepared for analysis using the EVOQ LC-MS/MS and the SCION GC-MS/MS (Bruker). The MRM method development workflow was set up using Compound Based Scanning. The target pesticides (Table 1) were selected from the software’s MRM library before being exported to the CBS method editor. The dwell time for each MRM is then automatically calculated based on its retention time window (timed MRM). A “built-in” processing method allows for easy updates of the retention times and method parameters and automatically updates qualitative and quantitative ion ratios based on the standards.

Excellent sensitivity was achieved for multi-residue pesticides in various vegetable matrices using both GC-MS/MS and LC-MS/MS systems. Examples of 1 and 5 parts per billion spiked samples in a spinach QuEChERS matrix analyzed by LC-MS/MS and GC-MS/MS are shown in Figures 2a and 2b. R-squared values show excellent linearity was achieved with each matrix.

In Conclusion

The monitoring of pesticides in fruits and vegetables is a key priority for international food safety. Many emerging export economies have substantially different regulations to their Western counterparts. This means stringent monitoring of pesticides is therefore essential to meet international regulatory requirements and ensure product safety. However in complex biological matrices, achieving the accuracy and robustness needed for these routine quality control methodologies can be complex and time consuming. Hardware and software developments in tandem mass spectrometry coupled chromatographic systems provide the sensitivity and selectivity needed for such routine operations, while reducing operator input and instrument down time, and simplifying method development.

Anacleto is VP Applied Markets at Bruker Daltonics. Reach him at joe.anacleto@bruker.com.

References Furnished Upon Request

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The Gulf of Mexico oil spill, the largest in U.S. history, has raised awareness of a food safety issue, namely contamination by polycyclic aromatic hydrocarbons (PAHs). In the future, analytical testing for PAHs in fish, crustaceans, and bivalves will undoubtedly become a routine procedure for many laboratories. PAH exposure, through either environmental pollution or contaminated foodstuffs, and its effects on human health have been the topic of many scientific studies. The recent oil spill again focuses attention on this toxic class of compounds.

PAHs comprise a large group of chemical compounds that are known cancer-causing agents. Some PAHs have been shown to be carcinogenic and mutagenic. The scope of monitored and regulated PAHs is under constant change, influenced by international advisory bodies such as the World Health Organization (WHO) and the European Food Safety Authority (EFSA). Changes in regulations highlight the need for more accurate quantification and improved detail in separation to isolate key PAHs from possible interfering isomers. Gas chromatography (GC), in combination with mass spectrometry (MS), is one of the principal analytical techniques used for identifying and quantifying PAHs in environmental and food-related samples.

During GC/MS analysis, some co-eluting PAHs exhibit an identical MS fragmentation pattern. The possible chromatographic co-elution of some PAHs therefore requires special attention. To obtain unambiguous identification and highly accurate quantification of priority and regulated PAHs, an optimized capillary column is essential.

Here we discuss the possibilities offered by a new generation of dedicated GC columns for PAH analysis, which contribute to more accurate reporting of these compounds for both food and environmental monitoring.

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Table 1. The scientific opinion of the panel on contaminants in the food chain on a request from the European Commission on Polycyclic Aromatic Hydrocarbons in Food (The EFSA Journal, 2008).

EPA Recommendations

Until recently, most analytical methods for PAH monitoring were established for analyzing the 16 priority pollutant PAH compounds recommended by the U.S. Environmental Protection Agency (EPA). They are naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[g,h,i]perylene, and indeno[1,2,3-cd]pyrene (see Table 1, p. 38). This list is often targeted for measurement in environmental samples.

The concern about environmental PAH pollution arises from the bioaccumulation risk in the food chain. PAH exposure occurs mainly through air inhalation and food consumption. Sources of airborne PAH include traffic and industry as well as tobacco smoke and open fires. Dietary exposure to PAHs through food consumption has recently gained importance because of general concern about food safety in the European Union and the United States. The oil spill in the Gulf will lead to mounting concern for food safety risks associated with marine organism consumption.

PAH occurrence in food is influenced by the same physiochemical characteristics that determine their absorption and distribution in man. Relative solubility in water and solvents, as well as volatility, determines their capacity for transport and distribution and, consequently, influences their uptake by living organisms. PAHs have a lipophilic nature that contributes to their accumulation in the lipid tissue of plants and animals. The waxy surfaces of vegetables and fruit can concentrate low molecular mass PAHs, mainly through surface absorption. The deposition of small PAH-contaminated airborne particles is the principle route of contamination for vegetables. Atmospheric fallout is also responsible for contamination of less volatile PAHs, which end up in fresh water or marine sediments. PAHs are strongly bound to these sediments, which then become potential reservoirs for PAH release. Filter feeding bivalves have a low metabolic capacity for PAHs, which may lead to their bioaccumulation. Oil spills are the other main cause of PAH contamination of marine organisms.

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Table 2. The most critical peak-pair separations for polycyclic aromatic hydrocarbons.

Smoking and Food Processing

Raw foods rarely contain substantial levels of PAHs, reflected by the relatively low-level background contamination in unprocessed foods from remote rural areas. The produce contamination level is already elevated in more populated regions because of airborne PAH emission by industry and motor vehicles.

Drying, smoking, grilling, roasting, and frying are major PAH-generating food processes and can contribute to alarming PAH levels. Smoked fish and barbequed meat may contain up to 200 µg/kg of PAHs. Charcoal-grilled duck breast steaks were reported to contain up to 300 µg/kg PAH. Smoke-processed duck breast steak contained up to 53 µg/kg of carcinogenic PAHs.

Vegetable oils used for seasoning and cooking, as well as those incorporated into biscuits and cakes, are significant dietary sources of PAHs. Their occurrence and level varies widely depending on drying processes and refining.

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Figure 1. Elution of critical PAH peak pairs on standard 5ms and 17ms-type GC columns.

In 2002, the former EU Scientific Committee on Food identified 15 PAHs as potentially carcinogenic and suggested benzo[a]pyrene as an indicator of the occurrence and effect of carcinogenic PAHs in food. In 2005, the Joint FAO/WHO Expert Committee on Food Additives confirmed these findings and proposed adding benzo[c]fluorine. This group of PAHs has become known as 15+1 EU priority PAHs (see Table 1, above).

Recent scientific opinion of the EFSA Scientific Panel on Contaminants in the Food Chain led to the adoption of an alternative and more limited list of PAHs for food risk characterization. Oral carcinogenicity data are only available for benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[g,h,i]perylene, chrysene, dibenzo[a,h]anthracene, and indeno[1,2,3-cd]pyrene.

For some time, benzo[a]pyrene was thought to be a suitable marker for the occurrence and effects of carcinogenic PAHs in food and was, therefore, the only regulated one. The EFSA panel concluded, however, that these eight PAHs (PAH8), either individually or in combination, were the best indicators of PAH toxicity in food. More recently, benzo[a]pyrene, chrysene, benz[a]anthracene, and benzo[b]fluoranthene (PAH4) have been suggested by the EFSA panel as suitable PAH indicators. The PAH4 EU regulation will come into effect in 2010-2011.

Accurate quantification of individual PAHs and PAH sets, either as 16 EPA, PAH(15+1), or the subset PAH4, requires separation of individual PAHs from interferences either by mass ion and/or by chromatographic separation.

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Figure 2. Critical PAH Peak-Pair Separations on the Varian Select PAH GC column, 30 m x 0.25 x 0.15 µm, Programmed GC/MS Analysis in SIM Mode.

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Figure 3. GC/MS analysis (total ion chromatogram [TIC]) of PAHs in salmon using a Select PAH column, 15 m x 0.15 mm x 0.10 µm.

GC/MS Analysis for PAH Monitoring

PAH analysis in food is typically performed using gas chromatography-mass spectrometry (GC/MS) operated in the selective ion-monitoring (SIM) mode. The SIM mode improves selectivity while increasing sensitivity. Given the rising number of PAH analytes targeted for routine monitoring, along with regulation changes and the fact that many PAH congeners exhibit identical MS ion fragmentation, the chromatographic separation and selectivity of the GC column used for PAH analysis has gained importance. Often, general-purpose GC columns do not deliver the degree of selectivity required by the new regulations for more detailed PAH analyses. The chromatographic separation of PAH congeners that have very similar chemical structures and molecular mass is challenging (see Table 2, left).

GC columns for PAH analysis should be able to distinguish the small structural differences that exist between PAHs with virtually similar physicochemical properties. The high-boiling nature of the 5/6-ring congeners requires high GC column elution temperatures, in excess of 325°C. Obviously, GC columns and liquid phases for the analysis of these 5/6-ring PAHs should therefore be highly temperature resistant.

Recently introduced columns based on ionic liquids lack the thermal robustness for elution of the 5/6 -ing PAHs and, hence, have a limited durability. The majority of liquid phases are based on polysiloxane backbone chemistry, which is generally more thermally resistant. In recent years, functional groups (i.e., phenyl) have been incorporated into the polysiloxane chain as arylene inclusions, increasing the thermal and oxidative resistance of the liquid phase. Columns coated with such phases can operate at higher temperatures. This increased thermal resistance is apparent at temperatures above about 300°C. These arylene low bleed columns support the elution temperatures necessary for high-boiling dibenzopyrenes.

Arylene- and phenyl-substituted liquid-phase columns provide a reasonable degree of selective PAH separation. The most popular PAH column choices are the non-polar 5% phenyl/arylene polysiloxane (DB-5ms, Rtx-5ms, VF-5ms, ZB-5ms) and mid-polar 50% phenyl/arylene polysiloxane phase columns (DB-17ms, Rtx-17ms, VF-17ms). However, both liquid phases will suffer from inaccurate quantification of key target EPA and EU PAHs due to co-eluting interferences such as benzo[j]fluoranthene and triphenylene.

Figure 1 (above) illustrates the separation and quantification difficulties for important priority EPA and EU PAHs. Chrysene cannot be measured accurately on non-polar or mid-polar columns because triphenylene co-elution may create biased results. In addition, benzo[b]fluoranthene cannot be quantified accurately on 5% phenyl/arylene columns due to co-elution with the benzo[j]fluoranthene isomer. Co-elution also occurs for the triplet indeno[1,2,3-cd]pyrene/benzo[b]triphenylene/dibenz[a,h]anthracene group on these 5% phenyl/arylene columns.

A new, selective GC column dedicated for PAH analysis, Varian Select PAH, was recently introduced that has the unique ability to isolate chrysene from the interfering triphenylene while simultaneously separating the three benzo[b,k,j]fluoranthene isomers. The liquid phase of this column incorporates highly selective selectors for PAH-isomer separation to overcome the limitations of other GC columns. The selective column can also separate other critical peak triplets such as indeno[1,2,3-cd]pyrene, benzo[b]triphenylene, and dibenz[a,h]anthracene (see Figure 2, below).

The improved separation characteristics of this column provide a more precise characterization of PAHs in various food matrices such as smoked haddock and salmon (see Figures 3-4, below and p. 42). Salmon was spiked with a mixture of EPA- and EU-regulated PAHs, as well as triphenylene as an important interference. The PAH concentration range varied from <0.5 parts per billion (ppb) up to 10 ppb for benzo[a]pyrene. For sample saponification, a potassium hydroxide in methanol solution was added to the homogenized and weighted salmon. After saponification, the PAHs were extracted with cyclohexane. The extract was concentrated and further cleaned using fully automated gel permeation chromatography directly coupled to an evaporation unit in the same analytical system. Dichloromethane was used as the eluent. The fraction containing the PAHs was concentrated and analyzed by GC/MS.

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Figure 4. Benz[a]anthrancene (BaA), cyclopenta[c,d]pyrene (CCP), chrysene (Chr), and interfering triphenylene (Tr), m/z 226 (blue), m/z 228 (red).

Fast, detailed analysis of PAH was obtained on a 15 m x 0.15 mm x 0.10 µm Select PAH column, with dibenzo[a,h]pyrene, the last PAH of interest, eluting at 28 min (see Figure 3, p. 40).

Chrysene and triphenylene were sufficiently separated to allow accurate chrysene quantification (see Figure 4, p. 42). Bias in chrysene quantification due to triphenylene interference has not been studied in detail in food matrices, mainly due their co-elution on most GC columns.

The higher molecular weight toxic dibenzopyrenes are usually less prevalent at low concentration levels. Dibenzopyrenes are prone to discrimination effects in the injector, and care must be taken to ensure complete evaporation to obtain higher responses. Their low volatility may also create adsorption effects in the MS interface and ion source.

Higher MS interface and ion-source temperatures can limit these phenomena, increasing responses and reducing peak tailing for these high molecular weight PAHs. Further significant enhancement of the analytical sensitivity for dibenzopyrenes, improvement of signal-to-noise (S/N) ratios, and faster elution can be achieved using thinner film columns of 0.1 – 0.15 µm as applied to the Select PAH column. The low-bleed performance and good S/N ratio of the Select PAH column at 350°C are apparent.

The presence of PAHs in vegetable oils is mostly related to contact with combustion gases from the seed drying processes. Refining methods such as deodorization and treatment with activated charcoal can reduce the PAH level significantly, but residues may remain.

Kuipers and Oostdijk are with Varian B.V., now part of Agilent Technologies, Middelburg, Netherlands. Schulz and Ruthenschroer are with Eurofins WEJ Contaminants GmbH, Hamburg, Germany. For more information, go to www.agilent.com or www.varianinc.com.

Resources

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• European Food Safety Authority (EFSA). Scientific opinion of the panel on contaminants in the food chain. EFSA J. 2008;724:1-114.
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