Achieving Highly Sensitive Dioxin Analysis with Novel GC–MS Methods

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Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) are toxic compounds categorized as persistent organic pollutants (POPs) and are ubiquitous throughout the world. Detecting trace levels of PCDD and PCDF is important to monitor food supplies and to ensure industrial emissions meet regulatory standards. In line with the ongoing innovation in dioxin analysis technology, the United States Environmental Protection Agency (US EPA) is currently evaluating a new method-atmospheric pressure gas chromatography tandem mass spectrometry (APGC–MS/MS)-for PCDD and PCDF confirmatory analysis. Joe Romano and Douglas Stevens from Waters Corporation discuss the benefits of this new method.

Persistent organic pollutants (POPs) are toxic compounds that are widespread throughout the environment. Organic substances characterized as POPs share a combination of physical and chemical properties, including long-term persistence in the environment, capability for long‑range transport, bioaccumulation in the food chain, and toxicity to humans and animals. Dioxins and furans belong to the group of POPs known as halogenated hydrocarbons, characterized by their low water solubility and high lipid solubility, which lead to the bioconcentration of these contaminants in tissue and their accumulation in the food chain. Chlorinated POPs, including pesticides such as dichlorodiphenyltrichloroethane (DDT), unintentionally produced dioxins and dioxin-like substances, and polychlorinated biphenyls (PCBs)-used in industrial applications-are all known to be damaging to human health and are restricted or banned under the 2001 Stockholm Convention on POPs.

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) arise through natural events, such as forest fires and volcanic eruptions, and anthropogenic routes, for example, as a by-product of industrial activities including waste incineration, decolorizing of paper pulp, and the manufacturing of some herbicides and pesticides.

Q. What are the negative health impacts of dioxins and dioxin-like substances and how is the reporting of data adapted to help better understand the potential toxicological impact of a contaminated sample?

Joe Romano: The hazardous effects of dioxins and dioxin-like substances to living organisms have been extensively studied and are well documented. They have been linked to a variety of conditions including type 2 diabetes, ischemic heart disease, and an acne-like skin disease called chloracne, which is a hallmark of dioxin exposure. The toxicity of PCDDs and PCDFs depends on the number and position of the chlorine atoms in the two benzene rings. Of the 210 congeners (75 PCDD congeners and 135 PCDF congeners), 17 possess a steric conformation that promotes their binding to the intracellular aryl hydrocarbon (Ah) receptor, which is known to mediate chemical toxicity.

Douglas Stevens: The toxic potential of the 17 congeners can be expressed relative to the most toxic compound, by the concept of toxic equivalence factor (TEF). The most toxic dioxin compound, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), is classified as a Group 1 carcinogen by the World Health Organization’s (WHO) International Agency for Research on Cancer (IARC) (1). This classification is based on evidence that 2,3,7,8-TCDD is a multi-site carcinogen in experimental animals and acts through a mechanism involving the Ah receptor.

Q. Why is the detection of these contaminants particularly challenging, and what steps have been taken to overcome this?

JR: Detecting trace levels of PCDD and PCDF is important to monitor food supplies, to ensure industrial emissions meet regulatory standards, to track sources of ongoing emissions, to identify contaminated sites, to inform risk assessments, and to contribute to human health assessment by measuring body burdens. However, as a result of their presence in trace amounts, highly sophisticated and sensitive analytical systems are required to measure them. These instruments require very low thresholds of detection, and are currently available only in a limited number of laboratories around the world. The analysis costs are also very high (2).

DS: Defining levels of tolerable exposure for preventative purposes is another challenge, and requires an assessment of current knowledge about the toxic effects of dioxins in animals and in humans. So improving understanding of how these compounds behave in the body, as well as developing increasingly sensitive analytical technology, are required to overcome the current challenges facing dioxin analysis.

Q. Can you briefly describe the technological improvements over the last five decades that have facilitated dioxin analysis?

DS: Prior to the 1970s, detection of dioxins was primarily obtained using packed‑column gas chromatography (GC) with electron capture detection (ECD) in the parts-per-million (ppm) and parts‑per‑billion (ppb) range (3). However, problems with specificity and detection limits, resulting from the technique’s inability to process highly complex environmental samples, spurred the development of gas chromatography–mass spectrometry (GC–MS) methods between the 1970–80s. Although GC–MS showed improvements and allowed many different sample types to be analyzed in order to determine the environmental distribution of dioxins, this method alone was insufficient and required intensive front‑end chemistry to separate trace analytes from potential interferences. Furthermore, during this period advances in both high performance liquid chromatography (HPLC) and high resolution capillary GC (HRGC) were applied to the separation of dioxin isomers. These developments were among some of the early steps aimed at enabling routine dioxin measurements to be conducted by relatively inexperienced analysts.

In the early days of highly specific and sensitive dioxin analytical methods, electron ionization (EI) GC–MS on a magnetic sector mass spectrometer
was the key technology used for highly precise, accurate, high-confidence quantitative environmental analysis, cementing its use for dioxin monitoring. However, despite the benefits of high resolution, magnetic sector instruments require a high degree of expertise and are expensive, limiting their widespread use. Current US EPA guidance recognizes HRGC/ high resolution mass spectrometry (HRMS) as the “gold standard” for dioxin and furan analysis in aqueous, solid, and tissue matrices under the EPA Method 1613B (4). However, GC–MS/MS gradually gained popularity and is now increasingly considered an equivalent method to the gold standard.

JR: In addition, the evolution of ionization methods has driven improvements in dioxin analysis. Atmospheric-pressure chemical ionization (APCI) sources with MS/MS offer a number of advantages over EI on a magnetic sector, including the capability for targeted analysis and very low detection of target analytes in complex samples. Additionally, EI is a hard ionization source, and extensive fragmentation associated with this technique can impact the abundance of the molecular ion and compound specific spectra. APCI is a softer technique, where the molecule is ionized by either proton transfer or charge transfer, rather than by direct electron bombardment, thus providing a more abundant molecular ion.

Q. How has the regulatory landscape driven analytical method development?

DS: In 2014, the European Union (EU) passed legislation recognizing the use of triple quadrupole GC–MS/MS, with either EI or APCI, as a confirmatory tool for checking compliance with maximum levels of dioxins in food and feed (589/2014/EU [5] and 709/2014 [6]). This was the result of an extensive validation study by an EU working group, and was the first official regulatory method that began the switch from magnetic sector technology and included APCI. The WHO conducted human-based risk assessments, setting TEFs for dioxins (7), where the results of dioxin analysis in samples should be reported as lower bound, medium bound, and upper bound concentration, by multiplying each congener by their respective TEF and summing them to provide the total concentration, expressed as toxic equivalency (TEQ). This system was developed to facilitate risk assessment and regulatory control of dioxins, furans, and PCBs.

JR: Following the 1999 dioxin crisis in Belgium (8) and the Stockholm Convention for POPs in 2001, the European Commission implemented an official continuous control strategy for food and feed. At the time, no standardized methods for food were available, but GC coupled with (magnetic sector)
GC–HRMS was widely accepted as the most sensitive and selective tool, especially in comparison with other MS analyzers such as time-of-flight (TOF) (9). The current US EPA guidance therefore recognizes HRGC/HRMS as the “gold standard” for dioxin and furan detection.

Q. A new method has been developed and is currently under evaluation by the US EPA. Can you explain how this method meets the requirements for dioxin analysis and its advantages over previous analytical approaches?

DS: In line with the ongoing innovation in dioxin analysis technology, the US EPA is currently evaluating a new method-APCI coupled with GC–MS/MS (APGC–MS/MS)-to develop and validate an alternative procedure for PCDD/PCDF analysis using MS/MS rather than HRMS. This method has already demonstrated that it is a robust and sensitive option for confirmatory analysis of PCDDs and PCDFs, in compliance with 589/2014/EU (10). The project aims to adapt Method 1613B protocols and criteria to MS/MS, showing equivalency of results in terms of sensitivity, linearity, selectivity, accuracy, and precision. Approval of a new method that uses GC–MS/MS for determining dioxins would take advantage of the technological advances made in tandem quadrupole MS over the decades, and also has the potential to lower laboratory costs.

JR: The GC–MS/MS method was developed and validated (Tier 3 validation). The atmospheric pressure source resulted in less fragmentation and therefore more sensitivity and selectivity than an EI source. The results of this method validation study, which are being submitted as an Alternate Test Procedure (ATP) to the US EPA, meet the Method 1613B specifications for PCDD and PCDF analysis, and method detection limit (MDL) and initial precision and recovery (IPR) test results showed excellent data quality (4).

Q. How will APCI combined with GC–MS/MS contribute to the continuing effort to ensure the safety of the global food chain?

DS: The upcoming method evaluation from the US EPA that will allow APGC–MS/MS as a confirmatory tool for checking compliance with minimum levels (MLs) of PCDD and PCDF will represent a key step in method development. Modern instrumentation is enabling laboratories in food, feed, and environmental industries to make increasingly sensitive measurements from complex samples, informing contamination incidences and allowing updated recommendations to be made on regulatory limits.

JR: Control measures within the food chain are needed to reduce human exposure to dioxins and furans, including air contamination and agricultural land and water. Today’s detection methods build on the early groundwork made in the 1970s and the subsequent optimizations made over the decades, and dioxin and furan research and detection methods have seen continuous improvement over the past 50 years. Compared with magnetic sector instruments, APGC–MS/MS has emerged as a more user-friendly technology that is likely to make dioxin analysis more accessible to testing laboratories across the world, therefore helping to secure global food safety.

References

1. IARC, Summaries & evaluations: Polychlorinated dibenzo-para-dioxins (Lyon, France, International Agency for Research on Cancer, p. 33, 1997 (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 69; http://www.inchem.org/documents/iarc/vol69/dioxin.html).
2. World Health Organization (WHO), Dioxins and their effects on human health factsheet (World Health Organization, [WHO], 2016). https://www.who.int/news-room/fact-sheets/detail/dioxins-and-their-effects-on-human-health.
3. R.E. Clement, American Chemical Society63(23), 1130–1139 (1991).
4. USEPA Region II Data Validation SOP for EPA, Method 1613 Revision B: Tetra- through Octa-Chlorinated Dioxins and Furans by Isotope Dilution HRGC/HRMS (December 2010).
5. Commission Regulation (EU) No 589/2014 of 2 June 2014 laying down methods of sampling and analysis for the control of levels of dioxins, dioxin-like PCBs and non-dioxin-like PCBs in certain foodstuffs and repealing Regulation (EU)No 252/2012, Offic. J. Eur. Commun. (2014), L164/18-40.
6. Commission Regulation (EU) No 709/2014 of 20 June 2014 amending Regulation (EC) No 152/2009 as regards the determination of the levels of dioxins and polychlorinated biphenyls, Offic. J. Eur. Commun. (2014), L188/1-18.
7. M. Van den Berg et al., The 2005 World Health Organization Re-evaluation of Human and Mammalian Toxic Equivalency Factors for Dioxins and Dioxin-like Compounds https://www.who.int/ipcs/assessment/tef_values.pdf.
8. N. Van Larebeke, L. Hens, P. Schepens, A. Covaci, J. Baeyens, K. Everaert, J.L. Bernheim, R. Vlietinck, and G. De Poorter, Environ Health Perspect.109, 265–273 (2001).
9. B. L’Homme, G. Scholl, G. Eppe, and J.F. Focant, Journal of Chromatography A1376, 149–158 (2015).
10. J. Dunstan et al., A Confirmatory Method for PCDDs and PCDFs in Compliance with EU Regulation 589/2014/EU Using Atmospheric Pressure Gas Chromatography (APGC) with Xevo TQ-S. Waters Application Notes No. 720005431en (June 2015).

Joe Romano is a senior manager, Food and Environmental Global Accounts, Waters Corporation in Milford, Massachusetts, USA.

Douglas Stevens is a principal scientist, Food and Environmental, Waters Corporation in Milford, Massachusetts, USA.

E-mail:Joe_Romano@waters.comWebsite: www.waters.com/apgc