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Advances in GC-MS/MS Enhance Routine Detection of Dioxins and Dioxin-like Compounds in Food and Animal Feed

By Richard Law
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Dioxins are highly toxic organic compounds that can remain in the environment for extended periods. These persistent organic pollutants (POPs), which include polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), are mainly generated by the combustion or manufacture of chlorine-containing materials such as plastics. Dioxins and other closely related POPs, such as polychlorinated biphenyls (PCBs), are classed as carcinogenic by the United States Environmental Protection Agency, and present a significant threat to human health even at low levels.

Dioxins and PCBs can enter the food chain when livestock consume contaminated animal feed, and can accumulate in the fatty tissues of animals due to their high fat-solubility. As a result, over 90% of human exposure to dioxins and PCBs is through the consumption of meat, fish, dairy and other foods of animal origin.1 Given the health risks posed by dioxins and PCBs, effective food testing workflows are essential to ensure products do not exceed regulatory-defined safe levels.

GC-MS/MS: A Robust Technique for Analyzing Dioxins and PCBs in Food and Animal Feed

To control human exposure to PCDDs, PCDFs and PCBs from the food chain, global regulatory bodies have established maximum levels (MLs) and action levels (ALs) for various POPs in food products, as well as approved analytical methods for food testing laboratories to enforce these standards. In the European Union (EU), for example, European Commission regulations 2017/644 and 2017/771 outline sampling, sample preparation and analysis protocols for the detection of dioxins and other dioxin-like compounds in food and animal feedstuffs.2,3

With food testing laboratories tasked with handling potentially hundreds of samples every day, these workflows must be supported by robust and reliable analytical technologies that can confidently identify and accurately quantify dioxins and PCBs with minimal maintenance requirements in order to minimize downtime and maximize throughput.

Thanks to ongoing improvements in the robustness and sensitivity of gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) systems, regulations were updated in 2014 to permit this technique as an alternative to gas chromatography-high resolution mass spectrometry (GC-HRMS) for confirmatory analysis and for the control of MLs and ALs. The latest GC-MS/MS systems are capable of exceptionally reliable performance for the routine analysis of dioxins and PCBs, providing accurate and sensitive quantification of these compounds even at trace levels.

Case Study: Sensitive and Reliable Determination of Dioxins Using GC-MS/MS

The performance of modern GC-MS/MS systems was evaluated in a recent study involving the confirmatory analysis and quantification of 17 PCDDs and PCDFs, and 18 dioxin-like and non-dioxin-like PCBs in solvent standards and various food and feedstuff samples. The samples were analyzed using a triple quadrupole GC-MS/MS system equipped with the advanced electron ionization source (AEI) and a TG-Dioxin capillary GC column. Two identical GC-MS/MS systems in two separate laboratories were used to assess the reproducibility of the method.

Extraction was performed by Twisselmann hot extraction or pressurized liquid extraction. The automated clean-up of the extracts was performed using a three-column setup, comprising multi-layered acidic silica, alumina and carbon columns. Two fractions were generated per sample (the first containing non-ortho PCBs, PCDDs and PCDFs, and the second containing mono-ortho and di-ortho PCBs and indicator PCBs) and these were analyzed separately. The analytical method gave excellent separation of all the PCDD, PCDF and PCB congeners in less than 45 minutes.

Given the high sensitivity of modern GC-MS/MS instruments, a calibration-based approach was used to determine limits of quantitation (LOQs) of the analytical system. Using calibration standards at the LOQ and subsequent check standards at this level enabled the performance of the method to be assessed throughout the analytical sequence. This also allowed LOQs for the individual congeners to be determined, assuming a fixed sample weight. Individual congener LOQs could be applied to upper-bound, middle-bound and lower-bound toxicity equivalence (TEQ) results by substituting the result of any congener that fell below the lowest calibration point with this value multiplied by the toxicity equivalence factor (TEF) of the congener.

To evaluate the response factor deviation over the course of the analytical sequences, standards at the specified LOQ were analyzed at the start, during and end of each run. Using a nominal weight of 2 g, and assuming 100% 13C-labeled standard recovery and all natives were less than the LOQ in the sample, a minimum upper-bound value of 0.152 pg/g WHO-PCDD/F-TEQ was determined. This met regulatory requirements for reporting at 1/5th of the ML upper-bound sum TEQ for all food and feedstuffs with a nominal intake of 2 g, with the exception of guidance associated with liver of terrestrial animals and food for infants or young children, which both have legal limits defined on a fresh weight basis. In these cases, either a larger sample intake or a magnetic sector instrument would be required. All of the calibration sequences demonstrated response factor %RSDs within EU regulations, highlighting the suitability of the method.

To demonstrate the performance of the GC-MS/MS system, six replicate extractions of a mixed fat quality control sample (QK1) were prepared, split between the two sites and analyzed at regular intervals throughout the analytical sequences (14 injections in total). The measured WHO-PCDD/F-TEQ values for congener were in excellent agreement with the reference value provided by the EU Reference Laboratory for Halogenated POPs in Feed and Food, and the upper bound WHO-PCDD/F-TEQ value did not deviate by more than 6% from the reference value for all 14 measurements (Figure 1). The deviation between the upper-bound and lower-bound WHO-PCDD/F-TEQ for each measurement was consistently less than 1.2%, well below the maximum limit of 20% necessary to support compliance with EU regulations.

pper- and lower-bound WHO-PCDD/F-TEQ values
Figure 1. Upper- and lower-bound WHO-PCDD/F-TEQ values for all 14 measurements of the QK1 mixed animal fat quality control sample, for six replicate extractions.

Robust Routine Analysis of Dioxin and Dioxin-like Compounds

To assess the robustness of the GC-MS/MS system, the PCDD, PCDF and non-ortho PCB extracts were pooled into a mixed matrix sample and analyzed more than 161 injection sequences across a period of approximately two weeks. Each sequence consisted of 40 matrix injections and 40 LOQ standards, interspersed with nonane blanks. No system maintenance, tuning or user intervention was undertaken throughout the two-week study. Figure 2 highlights the exceptional peak area stability achieved for selected PCDD and PCDF congeners.

Peak area repeatability
Figure 2. Absolute peak area repeatability over two weeks of analysis for selected PCDD and PCDF congeners in a pooled matrix sample (%RSD and amounts on column are shown for each congener).

These results highlight the exceptional levels of day-to-day measurement repeatability offered by the latest GC-MS/MS systems. By delivering consistently high performance without the need for extensive maintenance steps, modern GC-MS/MS systems are maximizing instrument uptime and increasing sample throughput for routine POP analysis workflows.

Conclusion

Developments in GC-MS/MS technology, namely the advanced electron ionization source, are pushing the limits of measurement sensitivity, repeatability and robustness to support the needs of routine dioxin and PCBs analysis in food and feed samples. By minimizing instrument downtime while maintaining exceptional levels of analytical performance, these advanced systems are helping high-throughput food testing laboratories to analyze more samples and ultimately better protect consumers from these harmful pollutants.

References

  1. Malisch, R. and Kotz, A. (2014) Dioxins and PCBs in feed and food – Review from European perspective. Sci Total Environ, 491, 2-10.
  2. European Commission. Commission Regulation (EU) 2017/644, Off J Eur Union, 2017, L92 9-34.
  3. European Commission. Commission Regulation (EU) 2017/771, Off J Eur Union, 2017, L115 22-42.

Acknowledgements

This article is based on research by Richard Law and Cristian Cojocariu (Thermo Fisher Scientific, Runcorn, UK), Alexander Schaechtele (EU Reference Laboratory for Halogenated POPs in Feed and Food, Freiburg, Germany), Amit Gujar (Thermo Fisher Scientific, Austin, US), and Jiangtao Xing (Thermo Fisher Scientific, Beijing, China).

Jeff Moore, USP
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Fighting the Reality of Food Fraud

By Jeff Moore, Ph.D.
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Jeff Moore, USP

Economically motivated adulteration (EMA) of food, or food fraud, has been estimated to cost the food industry $30–40 billion per year. The 2008 incident of melamine adulteration of milk powder has cost billions of dollars to companies and invaluable loss of consumer confidence. Even more significant than the economic cost or loss of confidence, the impact on public health was enormous. An estimated 290,000 consumers were affected with more than 50,000 hospitalizations including at least six deaths. There is also collateral damage caused by incidences of EMA, including the loss of confidence in government regulatory systems around food safety. Although major incidents like the melamine scandal happen infrequently, food fraud commonly occurs under the radar. According to a 2014 report by the Congressional Research Service, it is estimated that up to 10% of the food supply could be affected by food fraud Thus, the costs of fraud food are borne by industry, regulators and, ultimately, consumers.

Attend the Food Safety Supply Chain Conference, June 5–6, 2017 in Rockville, MD | LEARN MOREFood fraud is not a new phenomenon. During the time of the Roman Empire, Pliny the Elder wrote in Natural History about the adulteration of wine, bread and pepper, and tracked the fluctuation of their prices with the appearance of adulteration. In Medieval Germany, the adulteration of saffron was such a problem that the Safranschou Code was enacted, which described standards for saffron and allowed convicted adulterators to be executed.1 When there is an opportunity for economic gain, adulterators tend to come out of the woodwork.

As recently as the 1980s, food fraud was mostly an event confined to local markets. In 1981 the adulteration of olive oil with an industrial lubricant injured thousands and killed hundreds, but because the oil was not widely distributed, the primary effects were limited to Spain. Similarly, when apple juice adulteration occurred in the United States in the 1980s, the consequences were basically confined to the United States.

However, with the increasing globalization of the food supply chain and freer movement of foods and ingredients among countries, the opportunities for food fraud not only increased, but the consequences also now more easily have a global impact. By the late 1990s, the global consequences of food fraud became more evident with the contamination of fats intended for animal feed with industrial oils containing PCBs and dioxins. This scandal, which started with an oil recycler in Belgium, led to massive recalls of products throughout Europe and concerns about contaminated products reaching the United States. The impact of this episode arguably changed the food safety environment in Europe and led to the formation of the European Food Safety Authority. Likewise, the fallout from the adulteration of wheat gluten with melamine in 2008 likely contributed to the passage of new food safety legislation in the United States, including FSMA.

FDA has always acted against food fraud whenever there was an indication of public health hazards. With the passage of FSMA and the Preventive Controls for Human Food rule (published in September 2015), the agency has come full circle to its roots with Harvey W. Wiley, M.D. and his famous Poison Squad. Dr. Wiley formed his famous group to go after adulterators of foods. The Poison Squad was famously known for their willingness to consume suspect foods to test for adulteration. FDA’s history of Dr. Wiley states that “In the 1880s, when Wiley began his 50-year crusade for pure foods, America’s marketplace was flooded with poor, often harmful products. With almost no government controls, unscrupulous manufacturers tampered with products, substituting cheap ingredients for those represented on labels: Honey was diluted with glucose syrup; olive oil was made with cottonseed; and “soothing syrups” given to babies were laced with morphine. The country was ready for reform…” While the opportunities for fraud have not changed, luckily we no longer have to rely on human volunteers to detect adulterated food.

The new Preventive Controls rule published in September addresses EMA when there is a reasonable possibility that adulteration could result in a public health hazard. Companies are required to conduct a written hazard analysis, which should include hazards identification and evaluation. Companies are expected to identify “…known or reasonably foreseeable hazards that may be present in the food…The hazard may be intentionally introduced for the purposes of economic gain.”[i]  While companies were previously expected to be knowledgeable about microbiological hazards in their products, it appears that they now also have the responsibility to be knowledgeable about known or reasonably foreseeable hazards from EMA.

How can organizations identify potential EMA threats as part of hazards analysis? One way is via the Food Fraud Database, which is designed to help answer this question by taking a look into the past. Launched in 2012, the database provides the information necessary to identify ingredients with a past pattern or history of adulteration and the adulterants used—a perfect fit for the EMA requirement in FSMA. The database has more than 140,000 users from 194 countries documented.

After identifying an ingredient with a pattern/history of EMA, companies need to determine whether the ingredient may introduce potential food safety hazards and how to develop a control plan in response. To address those issues, USP undertook a project in 2013 to take a more holistic approach to identifying EMA vulnerable ingredients by looking at factors beyond history. It assembled a group of leading food adulteration experts to develop a first-of-its-kind guidance document that offers a framework for the food industry to help develop and implement preventive management systems to deal specifically with EMA.

The Food Fraud Mitigation Guidance became official in the Food Chemicals Codex (FCC) in September 2015, just as FSMA’s Preventive Rule for Human Food was published. The aim of the guidance is to assist manufacturers and regulators with identifying the ingredients most vulnerable to fraud in their supply chains and how to choose effective mitigation tools to combat EMA. This is a significant leap forward in the battle against food fraud—and a way to get ahead of criminals engaging in EMA. The guidance provides not only a solution to deal with FSMA’s EMA provision, but goes beyond FSMA to help organizations fulfill GFSI requirements to conduct a food fraud vulnerability assessment and control plan.

Thenadier (The innkeeper), in Les Miserables said in the lyrics of Master of the House:

“…

watering the wine and making up the weight

…

Food beyond compare. Food beyond belief

Mix it in a mincer and pretend it’s beef

Kidney of a horse, liver of a cat

Filling up the sausages with this and that”

While deceiving the unwary can seem humorous in fiction, in real-life food fraud can have extremely serious consequences to consumers and everyone involved with the production of safe food. There are multiple large-scale efforts in many regions and countries to address food fraud. The attention that is now focused on food fraud and the development of new tools such as Food Fraud Database cast a bright light that will hopefully make it more difficult for food fraudsters to operate.

Reference

  1. Willard, P. (2002), Secrets of Saffron: The Vagabond Life of the World’s Most Seductive Spice, Beacon Press, ISBN 978-0-8070-5009-5