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HomeChemical Analysis for Food and Beverage TestingAnalysis of Persistent Organic Pollutants in Fish and Milk

Analysis of Persistent Organic Pollutants in Fish and Milk

Katherine K. Stenerson, Analytical Sciences Liaison

Introduction

Persistent organic pollutants (POPs) are compounds that are not easily degraded by chemical, biological, or photolytic processes and thus persist in the environment for long periods of time. Many of these compounds were put into commercial use after World War II in manufacturing, agricultural, and other applications.  In addition to those that were specifically produced for the intended use, POPs also resulted from various industrial processes and the combustion of certain materials. An example of the latter is dioxins, which can be produced from waste incineration and the manufacture of paper and pulp.1 As designated by the Stockholm Convention, the POPs list originally consisted of 12 chemicals, including organochlorine pesticides, polychlorinated biphenyls (PCBs), and polychlorinated dibenzo-p-dioxins (PCDDs)/polychlorinated dibenzofurans (PCDFs). New compounds have since been added and these include additional organochlorine compounds, several organobromine compounds, perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), and chlorinated naphthalenes, and paraffins. New compounds under consideration for inclusion on the official POPs list are methoxychlor, the flame retardant Dechlorane Plus and the UV absorber UV-238.2 In addition to the compound classes previously mentioned, polynuclear aromatic hydrocarbons (PAHs) are also considered as POPs because of their lipophilicity and continuous presence in the environment.3

Since POPs are lipophilic, they will bioaccumulate in the fatty tissues of living organisms. Moving up the food chain, their concentration increases as they pass from one organism to another, a process known as “biomagnification”.1 As a result, POPs can end up in animal-based foods such as meat and milk. Exposure to these compounds has been associated with different toxic effects for humans, including cancer, immunotoxicity, developmental issues, and reproductive effects. For this reason, regulations on the limits of exposure via diet have been developed by various countries.

Various approaches exist for the extraction and analysis of POPs. In this work, the Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) sample preparation method developed by Anastassiades and Lehotay,4 was applied to two different food commodities: fish and milk. Specifically, QuEChERS was applied to the extraction and cleanup of PAHs from raw salmon and PCBs from cow’s milk. Fatty foods such as fish and milk are challenging matrices in that fats often get coextracted with the target analytes, causing interference and/or sensitivity issues during analysis. Two zirconia-based sorbents, Supel™ QuE Z-Sep and Z-Sep +, were evaluated for the cleanup of fatty samples. In work published by Sapozhnikova and Lehotay on extraction and analysis of POPs in catfish, it was found that Z-Sep was effective in background removal while obtaining good recovery and reproducibility.5 Z-Sep is proprietary zirconia-coated silica. Zirconia, with its empty d-orbitals, acts as an electron acceptor (Lewis Acid) and thus will bind molecules with electron-donating groups (Lewis Base). This includes the phosphate moiety in phospholipids and the OH groups in sterols, mono, and diglycerides, and the COOH group in free fatty acids. In the case of Z-Sep +, the silica particle is functionalized with both zirconia and C18. The purpose of the C18 is to act in a synergistic way in retaining hydrophobic molecules such as triglycerides that will not be removed by the zirconia. Analysis of the PCBs and PAHs was conducted by gas chromatography (GC) using different modes of detection; an electron capture detector (ECD) for the PCBs and mass spectrometry (MS) for the PAHs. The GC capillary columns used for both applications were specially selected to provide the necessary selectivity, temperature, and speed requirements.

EXPERIMENTAL

Samples of farm-raised salmon and whole cow’s milk were obtained at a local grocery store. For recovery studies, salmon was spiked with a mixture of 27 different PAHs and milk with 20 PCB congeners representing those most commonly occurring and the WHO-listed coplanars. The spiking levels were 100 ng/g for PAHs in salmon and 20 ng/g for PCB congeners in milk. QuEChERS extractions were conducted as described in Tables 1 and 2. The extracts were then subjected to cleanup with different sorbents per Table 3. The specified volume of extract was added to a tube containing the cleanup sorbent. The tube was then shaken for 1 min and centrifuged at 3400 rpm for 3 min. The resulting supernatant was analyzed by either GC-MS in selected ion mode (salmon extract for PAHs) or GC-ECD (milk extract for PCBs). The GC conditions for each analysis are listed in Tables 4 and 5. GC columns were specially selected to provide the necessary selectivity, temperature, and speed requirements. Specifically:

  • Salmon/PAH analysis: SPB-608, 20 m x 0.18 mm I.D., 0.18 µm (custom dimension) provided resolution of benzo [b], [j], [k] fluoranthene isomers and benzo [a] and [e] pyrene isomers. 
  • Milk/PCB analysis: The SLB-5ms, 20 m x 0.18 mm I.D., 0.18 µm provided a high maximum temperature for column “bake-out” of a heavy matrix, and with a hydrogen carrier, a faster analysis time than a standard 30 m column.
Table 1.Overview of QuEChERS extraction method used for PAHs in salmon.
Table 2.Overview of QuEChERS extraction method used for PCBs in cow’s milk.
Table 3.Cleanup sorbents used for salmon and milk extracts
Table 4.GC-MS conditions for the analysis of PAHs in salmon extract
Table 5.GC-ECD conditions of the analysis of PCBs in milk extract

Quantitation of the PAHs in the salmon extracts was done against a 5-point calibration curve prepared in solvent (5, 10, 20, 30, 50 ng/mL) with internal standard added to each level at 20 ng/mL. An uncleaned salmon extract was injected 3X prior to running the calibration standards and sample extracts. This was done to "prime" the GC-MS system and stabilize the response for the heavier PAHs. A 20 ng/mL check standard was run after each set of spiked samples to check instrument response.

Quantitation of the PCB congeners in the milk extracts was done against a 5-point calibration curve prepared in solvent (1, 5, 10, 20, 30 ng/mL). A 10 ng/mL check standard was run after each set of spiked samples to check instrument response. If a response drop was indicated, samples were re-run with a new calibration.

 

RESULTS AND DISCUSSION

Background Removal

GC-MS analysis in full scan mode was performed before and after cleanup on both sets of extracts. The effect of the cleanup varied with the matrix. In the case of salmon, there was a significant reduction in overall background for all cleanups (Figure 1), while with the milk there was a reduction only in the matrix peaks eluting after 15 minutes. This is shown for Z-Sep cleanup in Figure 2. In the salmon extracts, cleanups containing PSA showed peaks eluting in the first portion of the run, several of which coeluted with PAHs. This required blank subtraction in order to accurately quantitate the spiked samples. These peaks were most likely from contaminants bleeding off the PSA material. 

The Y-axis, represents the intensity of the signals in a GC-MS full scan, all plotted on the same scale. Each chromatogram corresponds to a different cleanup method applied to salmon extracts, with notable differences in peak patterns and intensities across the plots. The top chromatogram, in green, labeled "Z-Sep," shows relatively few peaks, indicating a clean extract. The second, in pink, labeled "PSA/C18," displays a moderate number of peaks with lower intensity compared to others. The third, in blue, labeled "Z-Sep+/PSA," reveals a dense cluster of peaks, particularly around the 8-minute mark, suggesting more retained compounds. The fourth chromatogram, in red, labeled "Z-Sep+," has prominent peaks at various retention times, with significant intensity near 6 and 8 minutes. The fifth chromatogram, in purple, labeled "Before cleanup," exhibits numerous, high-intensity peaks throughout, indicating a complex sample with little to no purification. The clear distinction between chromatograms highlights the varying effectiveness of the cleanup methods in removing interfering substance.

Figure 1.Comparison of salmon extracts before and after cleanup, GC-MS full scan (all are same Y-scale).

Two chromatograms comparing cow’s milk extract before and after cleanup using Z-Sep, plotted on a shared X-axis labeled "Time (min)" ranging from 10 to 45 minutes. The Y-axis, labeled "AU," represents absorbance units on a logarithmic scale from 1.00E+07 to 3.00E+07, consistent across both graphs. The upper chromatogram, labeled "Before cleanup," shows numerous sharp and intense peaks, particularly between 25 and 35 minutes, with the most prominent peaks reaching the top of the Y-axis at 3.00E+07. This indicates a highly complex mixture containing significant interfering compounds. The lower chromatogram, labeled "After cleanup," reveals a cleaner profile with fewer peaks and reduced intensity, the tallest peaks reaching approximately 1.65E+07. The reduction in peak density and height after cleanup highlights the effectiveness of Z-Sep in removing impurities and simplifying the sample matrix for GC-MS analysis.

Figure 2.Cow’s milk extract before and after cleanup with Z-Sep, GC-MS full scan (both are same Y-scale).

For both matrices, the Z-Sep cleanup resulted in the lowest background. After cleanup, all PAHs were easily detected using GC-MS/SIM from the spiked salmon samples; as shown in Figure 3 for Z-Sep. In the case of the milk extracts, background removal was evaluated by GC-MS, however, PCB analysis was performed by GC-ECD (Figure 4). This was done because the selective response of the ECD did not provide an adequate response to the compounds comprising the sample matrix, thus a difference in the background could not be discerned between cleaned and uncleaned extracts. It was also noted in the milk extracts that the clean-ups reduced the level of heavy, late eluting matrix, but did not totally eliminate it. This required a longer run time to "bake" it off the GC column. The use of backflush on the GC instrument could be used to eliminate this.

A chromatogram from a GC-MS/SIM analysis of polycyclic aromatic hydrocarbons (PAHs) extracted from salmon after Z-Sep cleanup. The X-axis represents time in minutes, spanning from 4 to 16 minutes, while the Y-axis indicates signal intensity without numerical labels. The chromatogram features numerous sharp, well-defined peaks of varying heights, signifying the detection of individual PAH compounds. Peaks are labeled sequentially with red numbers, ranging from 1 to 30, corresponding to distinct compounds. The most intense peak appears near the 6-minute mark, with other prominent peaks distributed across the time range. A few smaller peaks occur after 14 minutes, tapering off towards the end of the graph. Three closely spaced peaks around 12 minutes are highlighted with blue arrows, drawing attention to a separation of compounds.

Figure 3.GC-MS/SIM analysis of PAHs extracted from salmon after Z-Sep cleanup. The spiking level was 100 ng/g. (Peak IDs Table 6)

A chromatogram from a gas chromatography-electron capture detector (GC-ECD) analysis, depicting the separation of PCB congeners extracted from whole cow’s milk. The x-axis represents the time in minutes, ranging from 10.0 to 18.0 minutes, while the y-axis is unlabelled but typically represents detector response or signal intensity. The chromatogram features several sharp peaks of varying heights, each labeled with a number corresponding to specific PCB congeners. Notable peaks include those at approximately 10.5, 11.0, 12.3, 12.7, 13.0, 13.8, 14.0, 14.2, 14.5, 15.0, 15.3, and 17.2 minutes, among others. The background is white, and the peaks are represented by black lines, providing a stark contrast for clarity. The chromatogram is a visual representation of the analysis used to identify and quantify the presence of PCB congeners following a Z-Sep cleanup, with a spiking level of 20 ng/g in the sample.

Figure 4.GC-ECD analysis of PCB congeners (Table 7) extracted from whole cow’s milk, after Z-Sep cleanup. Spiking level of 20 ng/g.

Analyte Recoveries

Recoveries of the PAHs spiked into salmon are summarized in Figure 5 for the different cleanups. Three replicates of salmon extract were cleaned using each sorbent type, with the averages reflected in the graph. No PAHs were detected in the blank salmon samples. Repeatability for all cleanups was very good, with RSDs <10% for most PAHs and cleanups (with many <5% RSD). Overall, Z-Sep and Z-Sep+ exhibited the best recoveries. Between these two cleanups, Z-Sep+ extracts had recoveries of >80% for all analytes, while Z-Sep performed well for most PAHs (recovery >70%). A general trend of decreasing recovery with increasing molecular weight was noted for all cleanups. This could be due to the more limited solubility of heavier PAHs in the acetonitrile extraction solvent. A less polar solvent would most likely lessen this trend, however, it would undoubtedly co-extract more matrix which in turn may require more extensive cleanup and/or use of MS/MS for increased sensitivity and selectivity.

A line graph illustrating the average percentage recoveries of polycyclic aromatic hydrocarbons (PAHs) from salmon, based on three spiked replicates at a spiking level of 100 ng/g. The x-axis lists various PAH compounds, such as Naphthalene, Acenaphthylene, Fluoranthene, and Benzo(g,h,i)perylene, among others. The y-axis represents the average percentage recovery, ranging from 50% to 130%. The graph includes four colored lines representing different cleanup methods: Z-Sep+ in blue, Z-Sep+/PSA in red, Z-Sep in green, and PSA/C18 in purple. Each line fluctuates across the x-axis, showing variations in recovery percentages for each PAH. The lines intersect and diverge, indicating differences in recovery efficiency between the methods. Notably, some peaks exceed 100% recovery, while others fall below 80%, demonstrating variability in the process. The background is white, and the lines are distinct in color, with markers such as squares and triangles to differentiate the methods visually.

Figure 5.PAH recoveries from salmon, average of 3 spiked replicates (spiking level of 100 ng/g).

The recoveries of the PCBs from cow’s milk are shown in Figure 6. Overall, recoveries averaged in the range of 70-120% for a majority of the targeted congeners. It was noted that in general, recoveries declined with increasing molecular weight/degree of chlorination. Specifically, PCB congeners no. 157, 180, 169, 189, and 209 generally had recoveries <80%. Z-Sep showed a slight advantage over the other sorbents for recoveries of these congeners. Results between the different clean-up sorbents were comparable for the lighter congeners. Repeatability was very good for all clean-ups, with RSDs <10% for all but one congener/cleanup combination (congener no. 81 - Zep+/PSA cleanup).

 line graph displaying the average recoveries of polychlorinated biphenyls (PCBs) from whole cow’s milk, based on three spiked replicates at a spiking level of 20 ng/g. The x-axis lists various PCB congeners, labeled numerically from No. 28 to No. 209. The y-axis indicates average recovery percentages, ranging from 40% to 120%. The graph features four colored lines representing different cleanup methods: Z-Sep+ in blue, Z-Sep in red, PSA/C18 in green, and Z-Sep+/PSA in purple. Each line shows fluctuations across the x-axis, highlighting variations in recovery percentages for each PCB congener. The lines often intersect and diverge, indicating differences in recovery efficiency between the methods. Peaks are observed near the beginning of the graph, with recovery percentages exceeding 100% for some congeners, while other points drop below 80%, illustrating the variability in recovery rates. The background is white, and the distinct colors of the lines, along with markers such as squares and triangles, help differentiate the methods visually.

Figure 6.PCB recoveries from whole cow’s milk, average of 3 spiked replicates (spiking level of 20 ng/g).

CONCLUSION

QuEChERS extraction and cleanup were evaluated for the extraction of PAHs and PCBs from salmon and whole cow’s milk, respectively. For the cleanup step, different sorbents were evaluated. GC-MS data indicated that of the four cleanups, Z-Sep removed the most background in both the salmon and milk extracts. This agrees with the findings for catfish published by Sapozhnikova and Lehotay.5 For recoveries after cleanup, Z-Sep and Z-Sep+ yielded the best results for PAHs from salmon, while Z-Sep showed a slight advantage for PCB recovery from milk. In addition, the columns chosen for GC analysis offered advantages for these applications. The special dimension SPB®-608 provided resolution of PAH isomers and a faster analysis time. The SLB®-5ms provided a high maximum temperature for eluting heavy matrix, and the shorter length and narrow ID in combination with the hydrogen carrier provided for a faster analysis time while still achieving the required resolution.

Explore also the SLB®-ILPAH column on our ionic liquid GC column page, providing separation for difficult PAH isomer sets.

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REFERENCES

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Dec. 2009. Persistent Organic Pollutants: A Global Issue, A Global Response. [Internet]. Environmental Protection Agency (EPA).[cited 01 Mar 2021]. Available from: https://www.epa.gov/international-cooperation/persistent-organic-pollutants-global-issue-global-response#popsepa.gov
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Stockholm Convention on Persistent Organic Pollutants (POPs). [Internet]. Available from: https://www.pops.int/
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Anastassiades M, Lehotay SJ, Štajnbaher D, Schenck FJ. 2003. Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning and “Dispersive Solid-Phase Extraction” for the Determination of Pesticide Residues in Produce. J AOAC Int. 86(2):412-431. https://doi.org/10.1093/jaoac/86.2.412
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Sapozhnikova Y, Lehotay SJ. 2013. Multi-class, multi-residue analysis of pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers and novel flame retardants in fish using fast, low-pressure gas chromatography–tandem mass spectrometry. Analytica Chimica Acta. 75880-92. https://doi.org/10.1016/j.aca.2012.10.034
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