Analyzing Persistent and Emerging Contaminants in Food

Preventing environmental contaminants from getting in to the food chain is of paramount importance to us all. Yelena Sapozhnikova, a Research Chemist at the Agricultural Research Service, United States Department of Agriculture (USDA) in Wyndmoor, PA, USA, spoke to The Column about her research into the development and evaluation of analytical methods for persistent and emerging organic chemical contaminants in food samples.

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Q. You recently developed a rapid sample preparation and gas chromatography tandem mass spectrometry (GC–MS–MS) method for the analysis of pesticides and environmental contaminants in fish. Can you tell us how you developed this method?

A: We try to be proactive in identifying potential hazardous contaminants that are not under surveillance yet, but that may potentially cause adverse or chronic health problems. For example, the European Food Safety Authority (EFSA) scientific opinion on emerging and novel brominated flame retardants (FRs) indicated that because of the lack of available analytical techniques for brominated FRs, and, therefore, lack of information on their occurrence in foods, a risk characterization was not possible.¹ At the same time, some of the brominated FRs have been shown to be genotoxic and carcinogenic, while others were identified as bioaccumulative, requiring monitoring in the environment and foods. We tried to fill this gap by developing the method for the analysis of a wide range of diverse FRs along with other classes of persistent organic pollutants (POPs) and pesticides.

Our goal was to develop a new advantageous method for more than 200 contaminants in fish and seafood, including a diverse range of pesticides, and persistent and emerging environmental contaminants. Environmental contaminants and pesticides were previously analyzed by separate methods, requiring either a different sample preparation technique or an additional chromatographic run. Integrating these contaminants into a multi-class, multi-residue method allows for a faster, less expensive, and higher‑throughput analysis.    We selected pesticides from different classes: stable organochlorines, organophosphate insecticides, nitrogen‑containing herbicides, and pyrethroids. Polychlorinated biphenyl (PCB) congeners were chosen based on the World Health Organization (WHO) list,2 including dioxin-like PCB congeners; polybrominated diphenyl ether (PBDE) congeners were selected to represent the most common congeners used in consumer products including banned penta-, and octa- congeners. Polycyclic aromatic hydrocarbons (PAHs) were selected based on the US EPA list3 and included PAHs identified as carcinogenic. Novel FRs were selected based on the proposed lists of prioritized FRs for environmental risk assessment, and included chlorinated, brominated, and organophosphate chemicals.

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  The extraction method was based on “quick, easy, cheap, effective, rugged, and safe” (QuEChERS) with acetonitrile, which allows nonpolar and relatively polar contaminants to be extracted, while also decreasing the amounts of co-extractive fat compared to commonly used non‑polar solvents like hexane or ethyl acetate. A dispersive solid-phase extraction clean‑up (d-SPE) approach with a zirconium‑dioxide‑based sorbent provided ~70% of co‑extractive material removal, and resulted in cleaner extracts and greater robustness for the gas chromatography tandem mass spectrometry (GC–MS–MS) analysis, and also lower instrument maintenance and idle time. Low pressure vacuum outlet GC (LPGC) provided fast separation of more than 200 analytes and 12 internal standards in 10 min.7–9 The majority of contaminants had excellent recoveries, even at low spiking levels, making the method applicable for analysis at environmentally relevant concentrations. 
     

Q. What were the challenges you faced and how did you overcome them? What are the advantages of this approach compared to other methods? 

A: The task of identifying potentially hazardous but not yet monitored contaminants is a challenge in itself. This requires intensive research into the newest publications, different countries’ proposed regulations, scientific guidance panels, the contaminant’s chemical properties, and so on. Obtaining analytical standards for method development when they are not commercially available is another challenge. Creating an efficient and rugged method covering a large amount of contaminants from different classes with satisfactory method performance was possible by selecting acetonitrile as an extraction solvent, and zirconium-dioxide based sorbent for clean-up.    While both the sample preparation and the analytical run in our method was rapid, data analysis for more than 200 analytes generated an enormous  amount of data points for each sample, and data processing and review was a  bottleneck. This is a challenge we have  yet to overcome.    The advantages of our method lies in its simplicity, speed, low cost, and high throughput. By using this method, one analyst can prepare a batch of 12 pre‑homogenized samples in 1 h in a few simple steps. Using disposable polypropylene tubes for extraction and d-SPE clean-up, there is no glassware to clean afterwards - who likes washing and solvent rinsing glassware?    The instrumental analysis using LPGC takes only 10 min to run one sample for more than 200 analytes, plus 2 min for cooling and re‑equilibrating, which translates to 40 samples for each 8 h shift, or 120 samples for a 24 h cycle - this is a very rather high throughput! Remember that typical laboratories use conventional GC with 30–40 min runs.   In comparison with traditional methods for pesticides and POP analysis based on pressurized fluid extraction (PLE), gel permeation chromatography (GPC), solid‑phase extraction (SPE) clean-up, and conventional GC, our method is less expensive, faster in terms of both sample preparation and GC analysis time, and produces less hazardous organic solvent waste, which reduces the environmental impact.  

Q. In another study, you evaluated different variables affecting extractability of incurred contaminants in fish samples. Could you talk a little about this? What were your results?

A: When new analytical methods are developed and validated for contaminants in food or environmental matrices, samples are spiked (fortified) in the laboratory, and method performance is accessed based on the spiked samples. However, analytes are more easily extracted from laboratory-spiked samples than from incurred samples, in which analytes are incorporated into the matrix and have ber analyte-matrix interactions to overcome. Therefore extraction efficiency of spiked samples can be different from extraction efficiency of incurred samples. Accurate results for real samples depends on all aspects of the analytical process, including sample processing, but unfortunately, this part is often ignored during analytical method development and validation. Even after method implementation, quality control samples used to check ongoing method performance are typically spiked samples, not incurred. Standard reference materials (SRMs) with certified contaminant concentrations are an excellent means to determine true extractability, but most of the time SRMs are not available for many contaminants and sample types.    The goal of our study was to investigate variables impacting QuEChERS-based extraction yields of incurred pesticides and environmental contaminants in fish with different lipid content. The variables we assessed included sample size, sample/solvent ratio, extraction times, and extraction devices.

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  Our results showed that 2 g test portions (rather than 10–15 g used in typical QuEChERS extraction) were adequate for the analysis of the incurred contaminants. Smaller subsample size often translates into faster, easier, and less wasteful methods, as long as the test portion meaningfully represents the original sample. Reduced sample size and smaller amounts of organic solvents needed for extraction produce less organic solvent waste, leading to greener, more environmentally friendly methods. In terms of other variables, our results showed that 1 min extraction with the pulsed-vortexing shaker was sufficient for extraction of the 35 incurred contaminants detected in the fish.  

Q. In your view, what are the main challenges associated with the analysis of contaminants in food samples? 

A: In my opinion, we should pay more attention to emerging, often yet unrecognized contaminants. Many potential contaminants may be missed during regular targeted monitoring regardless of their levels of contamination and toxicity.   For decades, pesticides and persistent organic pollutants (POPs) have been monitored in the domestic and imported food supply to ensure safety of consumed food. However, other previously unrecognized contaminants have been emerging and have become a greater regulatory concern in food safety programmes as a result of their persistence in the environment, and ability to bioaccumulate in tissues and biomagnify in the food chain, as well as potential adverse effects on human health and the ecosystem. It is important that these previously unrecognized emerging contaminants are included among traditionally monitored chemicals in foods to provide risk assessment data for the better protection of human health and the environment.    I mentioned some challenges before, such as identifying potentially hazardous contaminants, and obtaining analytical standards for method development when the standards are not commercially available.    Trying to cover a wide range of contaminants with different properties in one uncomplicated high throughput method is not an easy task; finding the golden mean with all contaminants achieving satisfactory method performance can also be a daunting one.   Food composition complexity is another factor that complicates analysis. Co‑extractive materials from food samples (for example, lipids, fats, sugars, pigments, etc.) complicate the analysis, resulting in retention time shifts, matrix effects, and inaccurate results. We need to find better ways to produce cleaner extracts and account for matrix effects by using isotopically labelled internal standards, matrix-matched calibration curves, and analyte protectants in GC, all while keeping the analytical methods relatively simple, fast, and cost-efficient. There are plenty of challenges in food analysis to overcome, there is never a dull moment, and that is what makes it interesting and fun. 
   

Q. What are you currently working on?

A: We are currently modifying and validating the method we developed for fish and seafood for cattle, swine, and poultry. Emerging contaminants like FRs are not routinely monitored in meats, but they are large volume production chemicals with lipophilic properties, which have been detected in wastewater and sludge, marine mammals, and have also been reported to bioaccumulate in tissues.    We are also evaluating a fast high‑throughput flow injection analysis technique for rapid screening of contaminants in foods. This approach provides ultra-fast (1–2 min) screening for multiple contaminants.   Another project we are undertaking focuses on food packaging (FP) contaminants - chemicals migrating from FP materials into packaged foods. While these chemicals are often uncharacterized, they can potentially be hazardous,  leading to unintentional exposure of the consumer.    All of these projects have one common denominator - to develop effective technologies to enhance food safety control and protect public human health.  

Disclaimer: The views and opinions expressed in this interview are those of the author and do not necessarily reflect the views of USDA or U.S. government. 

References

1. European Food Safety Authority “Scientific Opinion on Emerging and Novel Brominated Flame Retardants (BFRs) in Food” (2012).
2. M. Van den Berg, L.S. Birnbaum, M. Denison, M. De Vito, W. Farland, M. Feeley, H. Fiedler, H. Hakansson, A. Hanberg, L. Haws, M. Rose, S. Safe, D. Schrenk, C. Tohyama, A. Tritscher, J. Tuomisto, M. Tysklind, N. Walker, and R.E. Peterson, Toxicological Sciences 93, 223–241 (2006).
3. United States Environmental Protection Agency. Office of Solid Waste, Polycyclic Aromatic Hydrocarbons (PAHs). http://www.epa.gov/osw/hazard/wastemin/priority.htm 
4. California Environmental Contaminant Biomonitoring Program (CECBP) Scientific Guidance Panel (SGP). Materials for the December 4-5, 2008 Meeting. Brominated and chlorinated organic chemical compounds used as flame retardants. http://oehha.ca.gov/multimedia/biomon/pdf/120408flamedoc.pdf 
5. P.R. Fisk, A.E. Girling, R.J. Wildey. Prioritisation of flame retardants for environmental risk assessment. 2003 https://www.gov.uk/government/uploads/system/uploads/attachment_data/fil... (accessed 1 July 2012)
6. EFSA Panel on Contaminants in the Food Chain (CONTAM). Scientific Opinion on Polybrominated Biphenyls (PBBs) in Food. 8 (10) (2010) 1789. http://www.efsa.europa.eu/en/scdocs/doc/1789.pdf
7. Y. Sapozhnikova, Journal of Agricultural and Food Chemistry 62, 3684–3689 (2014).
8. Y. Sapozhnikova, LCGC North America32, 878–886 (2014).
9. Y. Sapozhnikova and S.J. Lehotay, Analytica Chimica Acta758, 80–92 (2013).
10. Y. Sapozhnikova and S.J. Lehotay, Journal of Agricultural and Food Chemistry63, 5163–5168 (2015).

Yelena Sapozhnikova

is a Research Chemist at the Agricultural Research Service, United States Department of Agriculture (USDA) in Wyndmoor, PA, USA. Dr. Sapozhnikova’s research focuses on the development and evaluation of new, advantageous analytical methods for persistent and emerging organic chemical contaminants in food and environmental samples. Her research involves improving all aspects of sample processing, preparation, clean-up, and chromatographic separation with mass spectrometry detection to make the analysis more efficient, fast, and cost‑effective. Dr. Sapozhnikova has developed novel methods for analysis of pesticides, diverse environmental contaminants, environmental estrogens, flame retardants, synthetic musk fragrances, pharmaceutical and personal care products, and other emerging contaminants to improve food and environmental safety and reduce health  risk factors.

E-mail: yelena.sapozhnikova@ars.usda.gov

Website: http://www.ars.usda.gov/pandp/people/people.htm?personid=47132

This article was featured in LCGC's digital magazine, The Column. Click here to view the full issue>>