Using Analytical Techniques to Follow Standards for Seafood Safety

Polychlorinated Biphenyls (PCBs) include a group of chlorinated compounds with over 200 variations, or congeners, with distinct chemical and physical characteristics. PCBs can be released into the general environment through many sources. For example, by illegal or improper dumping of PCB wastes, such as transformer fluids, from poorly maintained toxic waste sites, by disposal of PCB-containing consumer products in municipal landfills, and through leaks or fugitive emissions from electrical transformers whose oil often contains PCBs.

Chronic exposure of PCBs to animals can result in hormone balance disruptions, cancer, or reproductive failures. A key source of human PCB exposure can be foods, usually from fish and animal fat (Table 1). PCBs are lipophilic, and they preferentially separate from water and adsorb onto sediment at the lake and river beds. Bottom feeders and other aquatic organisms subsequently consume and accumulate PCBs, leading to a bio-concentration effect, which moves upward in the food chain.

Table 1. Maximum level for the sum of the six targeted PCBs and limit of quantitation per congener in seafood.

Foodstuff Maximum level Limit of Quantitation
Sum of PCB 28, 52, 101, 138, 153, 180
Muscle meat of fish, shellfish, and products thereof 75 ng/g wet weight 1 ng/g per congener

 

The European Commission Regulations EU 252/2012[1] and 1259/2011[2] classify PCBs into dioxin-like (DL-PCB) and non-dioxin-like PCBs (NDL-PCB) on the basis of their structural characteristics and toxicity, thus resulting in various technologies and maximum levels for these two groups. The technique described in this article includes six marker NDL-PCBs: PCB 28, 52, 101, 138, 153, and 180.

These PCBs constitute around half of the total amount of NDL-PCBs found in foodstuffs, and their sum is regarded as an ideal marker for occurrence and human exposure to NDL-PCBs and is thus set as the maximum level.[2] Performance standards for analysis of NDL-PCBs by GC-MS/MS are elaborated within the EU 589/2014 regulations.[4]

Polycyclic aromatic hydrocarbons (PAHs) are powerful atmospheric pollutants and are of concern since some have been identified as mutagenic, carcinogenic, or teratogenic. Just like PCBs, PAHs are lipophilic and usually have a very poor aqueous solubility. Thus, they can build up in lipid tissues of animals and plants. PAHs present in air (by deposition), soil (by transfer), or water (deposition and transfer) can contaminate foods. Some PAHs are semi-volatile; however, the majority of them tend to adsorb on organic particulate matter. Heavier PAHs preferably combine with particulate matter; therefore, a major route of contamination is atmospheric fallout. When particulates fall out onto a water surface, they are transported in suspension, ultimately ending up in freshwater or marine sediments.

PAHs form a strong adherence with these sediments, efficiently developing a potential pollution reservoir for subsequent PAH emission. Sediment-dwelling and filtering organisms are the most vulnerable to contamination. A majority of the organisms have a high bio-transformation capacity for PAHs; thus, there is no important bio-magnification in the aquatic food chain. However, filter-feeding bivalves, such as mussels and oysters, may accumulate PAHs since they filter large volumes of water and have a low metabolic potential for these compounds.

Until 2008, benzo(a)pyrene was employed as a marker for the occurrence of PAHs in foods. However, in 2008, the Scientific Panel on Contaminants in the Food Chain of the European Food Safety Authority (EFSA) inferred that benzo(a)pyrene alone was not an ideal marker for the occurrence of PAHs in foods and that a system of four specific substances (benzo(a)anthracene, benzo(a) pyrene, benzo(b) fluoranthene, and chrysene) would be more ideal markers.[4] As a result, Commission Regulation EU 835/2011[5] amended Regulation (EC) 1881/2006 in order to set maximum levels in particular foodstuffs for the sum of these four PAHs, while Commission Regulation EU 836/2011[6] set their LODs and limits of quantitation (LOQs), as represented in Table 2.

Table 2. Maximum levels for benzo(a)pyrene and the sum of the four PAHs and limits of detection and quantitation.

Foodstuff Maximum level (μg/kg) Limit of Detection Limit of Quantitation
Benzo(a) pyrene Sum of 4 PAHs
Smoked sprats and canned smoked sprats, bivalve mollusks (fresh, chilled or frozen), heat treated meat and heat treated meat products sold to the final consumer 5.0 30.0 ≤ 0.30 μg/kg for each of the four substances ≤ 0.0 μg/kg for each of the four sub-stances
Bivalve mollusks (smoked) 6.0 35.0

 

A technique has been devised for the concurrent analysis of the six markers NDL-PCBs and 16 PAHs (including the four specifically regulated PAHs) in bivalve mollusk samples with the help of the Bruker EVOQ GC-TQ Premium MS/MS system.

Experiment

Sample Preparation

Mussel and clam samples were gathered from the southern coast of Spain. Samples (8 g) were hydrolyzed with MeOH and KOH, filtered, and then extracted with n-hexane. The extracts were loaded into a cartridge containing alumina and florisil[7–9] in order to purify them. The purified extracts were evaporated to dryness under a nitrogen stream and reconstituted in 2 mL of cyclohexane:toluene (9:1). The sample preparation workflow is illustrated in Figure 1.

Sample preparation workflow.Figure 1. Sample preparation workflow.

PCB and PAH standards were collected from Dr Ehrenstorfer GmbH (Augsburg, Germany) and spiked samples were prepared.

Table 3. Mass spectrometry method conditions

Mass Spectrometer Bruker EVOQ GC-TQ MS system
MS Conditions
Ionization EI, 70 eV
Emission Current 40 μA
Active Focusing Q0 135 °C with Helium
Transfer Line Temperature 300 °C
Source Temperature 300 °C
CID Gas Ar, 2.0 mTorr
Detector Mode EDR
Scan Mode MRM, 0.6 sec/scan
Gas Chromatograph Bruker 436 GC
GC Conditions
Injector 1177 Split/splitless
Sample Volume/Injection Mode 1 μL, splitless
Injector Insert 4 mm single taper splitless with deact. wool (p/n: SG092003)
GC Oven Temperature 70 °C (1.7 min)→ 30 °C/min→ 180 °C (0´)→ 5 °C/min→ 320 °C (17´)
GC Column Bruker BR-PCB, 40 m x 0.18 mm, 0.18 micron (p/n: BR58697)
Carrier Gas Helium, 0.8 mL/min constant flow
Total Run Time 50 min
Autosampler Bruker 8400 autosampler
Software Bruker MSWS 8.2.1/TASQ 1.4 processing software

 

Methodology

In total, 41 compounds were analyzed which includes 16 PAHs, 16 deuterated PAHs used as internal standards (IS) and 9 PCBs.

Up to three MRM transitions per compound were employed wherever possible in order to increase specificity. Table 4 gives a complete list of MRM transitions.

Table 4. MRM conditions for the PCBs and PAHs monitored

Compound Name RT (min.) Precursor Ion Quan Ion CE Confirm Ion 1 CE Confirm Ion 2 CE
Naphthalene-d8 6.61 136 134   132 -25 - -
Naphthalene 6.70 128 102   126 -20 127 -5
Acenaphthalene-d8 10.75 160 158   156 -25 - -
Acenaphthalene 10.80 152 150   151 -15 126 -28
Acenaphthene-d10 11.14 164 160   162 -18 - -
Acenaphthene 11.24 153 127   151 -25 152 -20
Fluorene-d10 12.84 174 172   170 -30 - -
Fluorene 12.95 165 164   163 -30 139 -30
Phenanthrene-d10 16.67 188 184   186 -20 - -
Phenanthrene 16.79 178 176   177 -10 152 -25
Anthracene-d10 16.98 188 184   186 -20 - -
Anthracene 17.08 178 176   152 -25 177 -10
PCB-28 15.30 256 186   151 -50 - -
PCB-30 18.01 256 186   151 -50 - -
PCB-52 18.94 292 222   257 -15 - -
PCB-101 22.21 326 256   291 -15 - -
Fluoranthene-d10 22.34 212 208   210 -15 - -
Fluoranthene 22.44 202 200   201 -15 152 -32
Pyrene-d10 23.53 212 208   210 -15 - -
Pyrene 23.63 202 200   201 -15 151 -45
PCB-153 25.43 360 290   325 -15 - -
PCB-138 26.61 360 290   325 -15 - -
PCB-183 27.11 394 324   359 -15 - -
PCB-180 29.04 394 324   359 -15 - -
Benzo(a)anthracene-d12 29.60 240 236   238 -20 - -
Benzo(a)anthracene 29.73 228 226   202 -30 227 -18
Chrysene-d12 29.82 240 236   238 -20 - -
Chrysene 29.96 228 226   202 -25 227 -18
PCB-170 30.22 394 324 32 359 -15 - -
Benzo(b)fluoranthene-d12 35.06 264 260   262 -30 - -
Benzo(b)fluoranthene 35.21 252 250   248 -60 224 -55
Benzo(k)fluoranthene-d12 35.16 264 260   262 -30 - -
Benzo(k)fluoranthene 35.30 252 250   248 -60 224 -55
Benzo(a)pyrene-d12 37.06 264 260   262 -30 - -
Benzo(a)pyrene 37.24 252 250   248 -60 224 -55
Dibenzo(a,h)anthracene-d14 45.10 292 288   290 -20 - -
Dibenzo(a,h)anthracene 45.50 278 276   250 -50 277 -20
Indene(1,2,3-c,d)pyrene-d12 45.45 288 284   286 -20 - -
Indene(1,2,3-c,d)pyrene 45.75 276 272   273 -45 274 -40
Benzo(g,h,i)perylene-d12 48.16 288 286   284 -40
Benzo(g,h,i)perylene 48.47 276 272   273 -45 274 -40

 

Results and Discussion

The procedures and analytical demands within the EU[1–6] to monitor the levels of PAHs and NDL-PCBs in foodstuffs are very stringent and must conform to performance criteria with regards to linearity, accuracy, and precision, among other criteria. As per the provisions of the EU regulations, laboratories shall be accredited following the ISO 17025 standard by a standard body operating according to ISO Guide 58 to guarantee the analytical quality assurance. The results demonstrated below include all the analytical quality criteria demanded by the European regulations.

GC Separation

The GC operating conditions were optimized to achieve an optimal peak shape without any tailing effects, specifically for the late eluting PAHs as demonstrated in Figure 2. Furthermore, this optimized separation offered high sensitivity.

Total Ion Chromatogram (TIC) of 12.5 ppb standard mix (PAHs and PCBs).

Figure 2. Total Ion Chromatogram (TIC) of 12.5 ppb standard mix (PAHs and PCBs).

An exceptional chromatographic separation for the more critical pairs of compounds (as shown in Figure 3) could be obtained with the help of a narrow bore capillary column (40 m x 0.18 mm), thereby eliminating peak co-elution that could potentially mask some peaks and yield erroneous outcomes.

Total Ion Chromatogram (TIC) of 12.5 ppb standard mix (PAHs and PCBs) expanded in indicated areas.

Figure 3. Total Ion Chromatogram (TIC) of 12.5 ppb standard mix (PAHs and PCBs) expanded in indicated areas.

Linearity

The linearity of response of this technique has been assessed from the reporting limits upward. Nine various solutions of increasing concentrations were made: 0.5 ppb, 1 ppb, 2.5 ppb, 5 ppb, 12.5 ppb, 25 ppb, 50 ppb, 75 ppb, and 100 ppb, and spiked with the same amount of deuterated standards. Each standard solution was examined in triplicate.

Figures 4 and 5 show selected calibration curves for PAHs and PCBs, respectively.

Calibration curves for selected PAHs from 0.5 ppb to 100 ppb.

Figure 4. Calibration curves for selected PAHs from 0.5 ppb to 100 ppb.

Calibration curves for selected PCBs from 0.5 ppb to 100 ppb

Figure 5. Calibration curves for selected PCBs from 0.5 ppb to 100 ppb

Illustrated in Table 5 is a review of the calibration results demonstrating the linearity of the method with regression coefficients R2 > 0.995 and relative standard deviation (RSD)

Table 5. Summary of calibration results, with nine calibration levels from 0.5 to 100 ppb

Compound name RSD (%) Compound name RSD (%)
Naphthalene 0.99995 8.88 PCB-138 0.99886 14.51
Acenaphthalene 0.99934 9.18 PCB-183 0.99558 14.51
Acenaphthene 0.99620 11.42 PCB-180 0.99785 10.50
Fluorene 0.99978 11.13 Benzo(a)anthracene 0.99956 12.7
Phenanthrene 0.99886 6.32 Chrysene 0.99959 12.06
Anthracene 0.99845 12.03 PCB-170 0.99613 14.94
PCB-28 0.99989 1.86 Benzo(b)fluoranthene 0.99854 7.90
PCB-30 0.99931 8.57 Benzo(k)fluoranthene 0.99674 11.12
PCB-52 0.99982 4.83 Benzo(a)pyrene 0.99721 13.78
PCB-101 0.99936 8.31 Dibenzo(a,h)anthracene 0.99613 14.40
Fluoranthene 0.99896 15.11 Indene(1,2,3-c,d)pyrene 0.99752 12.88
Pyrene 0.99855 7.50 Benzo(g,h,i)perylene 0.99780 16.39
PCB-153 0.99959 8.09 - - -

 

Sensitivity and Detection Limits

One standard solution with a concentration of 0.1 ppb (100 femtogram on-column) was injected three times in order to validate the sensitivity of the method. Signal-to-noise (S/N) ratio above 40 was obtained for all compounds and replicates. Thus, the LOD for all analytes is less than 0.1 ppb.

Figures 6 and 7 show the MRM chromatograms for selected PAHs and PCBs, respectively.

MRM chromatograms for selected PAHs at 0.1 ppb level (100 femtogram on-column).

Figure 6. MRM chromatograms for selected PAHs at 0.1 ppb level (100 femtogram on-column).

MRM chromatograms for selected PCBs at 0.1 ppb level (100 femtogram on-column).

Figure 7. MRM chromatograms for selected PCBs at 0.1 ppb level (100 femtogram on-column).

Precision and Repeatability

The precision, expressed as repeatability, was estimated on the results obtained from three replicate analyses of a mussel extract spiked with PAHs and PCBs at 0.8 µg/kg. It is to be noted that this level is somewhat below the LOQ (0.9 µg/kg) needed for PAHs (as shown in Table 2).

Figure 8 shows an example of repeatability for chosen PAHs and PCBs.

Three replicate injections of a mussel extract spiked with 0.8 µg/kg of PAHs and PCBs.

Figure 8. Three replicate injections of a mussel extract spiked with 0.8 µg/kg of PAHs and PCBs.

Outstanding relative standard deviation of less than 4% was obtained for all analytes in mussel extract spiked at 0.8 µg/kg, as represented in Table 6.

Table 6. Summary of area repeatability for selected PCBs and PAHs in a mussel extract spiked at 0.8 µg/kg

Compound name Replicate 1 (Area) Replicate 2 (Area) Replicate 3 (Area) Average (Area) RSD (%)
Acenaphthene 50042 50822 50083 50316 0.7
Acenaphthalene 10673 10596 10669 10646 0.3
Anthracene 44029 43066 43873 43656 1.0
Benzo(a)pyrene 23148 23450 23986 23528 1.5
Benzo(a)anthracene 43193 43780 44175 43716 0.9
Benzo(b)fluoranthene 261459 266883 261520 263287 1.0
Benzo(g,h,i)perylene 12876 12977 13155 13003 0.9
Benzo(k)fluoranthene 29562 29539 29145 29415 0.7
Chrysene 42581 43704 43343 43209 1.1
Dibenzo(ah)anthracene 13906 13937 14000 13948 0.3
Phenanthrene 47378 46220 47637 47078 1.3
Fluoranthene 46342 45928 45007 45759 1.2
Fluorene 41185 40994 42628 41602 1.8
Indene(1,2,3-c,d)pyrene 11494 11184 11072 11250 1.6
Naphthalene 21826 22009 22823 22219 2.0
PCB 101 53189 49522 53118 51943 3.3
PCB 138 40578 40392 40627 40532 0.2
PCB 153 141830 137724 135958 138504 1.8
PCB 180 47887 46108 47638 47211 1.7
PCB 28 33700 33934 32112 33249 2.4
PCB 52 32109 31240 31233 31527 1.3
Pyrene 74599 73822 73394 73938 0.7

 

Selectivity, Ion Ratios Stability, and Robustness

The response of analytes in spiked mussel samples was compared with those of spiked standards to test the selectivity. This study did not find any interferences or co-elution effects. Also, there was no deviation in retention times between samples and standard chromatograms. Stability of the ion ratio is also essential for an unequivocal identification when using triple quadrupole instruments, since it helps to avoid any false positive reporting.

A comparison of pyrene analysis in a wedge clam extract and standard solution is illustrated in Figure 9. The relative retention time (RRT) difference for pyrene in wedge clam extract and standard is −0.03%, while the tolerance permitted is ±0.25%.[4] For confirmation ion 1, the ion ratios difference is 1.1% and accepted tolerance is ±20%, and for confirmation ion 2, the ion ratios difference is 7.8% and accepted tolerance is ±50%.[4]

Analysis of a wedge clam extract (left) in comparison to a pyrene standard solution (right). Orange: Quantitation ion; Sky Blue: Confirmation ion 1; Dark Blue: Confirmation ion 2.

Figure 9. Analysis of a wedge clam extract (left) in comparison to a pyrene standard solution (right). Orange: Quantitation ion; Sky Blue: Confirmation ion 1; Dark Blue: Confirmation ion 2.

The robustness of the technique was confirmed by analyzing replicates of bivalve mollusks spiked with PAHs and PCBs; in particular, wedge and hard clams (referred to as berberechos and almejas respectively, in Spain). Wedge clam extracts spiked at 0.8 µg/kg with PAHs and PCBs are shown in Figures 10 and 11, respectively. As represented, all analytes are perfectly identified, exhibiting exceptional response for the quantitation ion, as well as the confirmation ions for an unequivocal identification, thereby avoiding any false positive identification and reporting. Reducing the amount of matrix content injected is an important criterion for evaluation, as it usually results in a decrease in the maintenance needed to maintain robustness and sensitivity.

Analysis of different wedge clam extracts spiked at 0.8 µg/kg PAHs. Each time window shows the MRM transitions used for each compound.

Figure 10. Analysis of different wedge clam extracts spiked at 0.8 µg/kg PAHs. Each time window shows the MRM transitions used for each compound.

Analysis of different wedge clam extracts spiked at 0.8 µg/kg PCBs. Each time window shows the MRM transitions used for each compound.

Figure 11. Analysis of different wedge clam extracts spiked at 0.8 µg/kg PCBs. Each time window shows the MRM transitions used for each compound.

To assess the performance of the instrument with a more diluted sample, hard clam extracts spiked at slightly below the requisite limit of quantitation (LOQ) for PAHs/PCBs (0.8 μg/kg) were diluted twice. The required LODs are still surpassed even after diluting the samples twice, as represented in Figure 12. This promotes an increase in robustness of the method as well as the instrument as per the European regulations for routine 24/7 operation.

Analysis of a hard clam extract spiked at 0.8 µg/kg with PAHs/PCBs and diluted two-fold (400 femtogram on-column). Each time window shows the MRM transitions used for each compound.

Figure 12. Analysis of a hard clam extract spiked at 0.8 µg/kg with PAHs/PCBs and diluted two-fold (400 femtogram on-column). Each time window shows the MRM transitions used for each compound.

Conclusion

A technique for the analysis of 16 PAHs and 6 markers NDL-PCBs by GC-MS/MS in bivalve mollusks has been devised as per the European Regulations. The outstanding selectivity, sensitivity, and robustness of the Bruker EVOQ GC-TQ Premium MS system allows limits of detection of <0.1 µg/kg while injecting only 1 µL of sample. This sensitivity enables working with diluted samples, which may extend the instrument cleaning and maintenance cycles. The fast 40 m x 0.18 mm GC column exhibits good resolution for compounds that usually co-elute (for example, PCB28/PCB31, B(b)F/B(k)F, B(a)A/Chrysene/Triphenylene).

In addition, the run time is decreased significantly when compared to 60 m columns. A broad linear calibration range (from 0.5 to 100 ppb) with R2 > 0.99 and RSD < 15% was achieved for all the analyzed compounds. The significant reproducibility and performance of the Bruker EVOQ™ GC-TQ MS yielded RSD (%) below 4% at the limit of quantitation for all the compounds analyzed in the seafood samples. This method can be validated for routine 24/7 operation if needed.

References

[1] Commission regulation EU No 252/2012. Laying down methods of sampling and analysis for the official control of levels of dioxins, dioxin like PCBs and non-dioxin-like PCBs in certain foodstuffs and repealing Regulation (EC) No 1883/2006.

[2] Commission regulation EU No 1259/2011 amending regulation (EC) No 1881/2006 as regards maximum levels for dioxins, dioxinlike PCBs and non-dioxin-like PCBs in foodstuffs.

[3] Scientific report of EFSA, Results of monitoring of non-dioxin-like PCBs in food and feed, EFSA Journal OR European Food Safety Authority Journal 2010; 8(7):1071.

[4] Commission regulation (EU) No 835/2011 amending regulation (EC) No 1881/2006 as regards maximum levels for polycyclic aromatic hydrocarbons in foodstuffs.

[5] Commission regulation (EU) No 836/2011 amending regulation (EC) No 333/2007 laying down the methods of sampling and analysis for the official control of the level of lead, cadmium, mercury, inorganic tin, 3-MCPD and benzo(a)pyrene in foodstuffs.

[6] NOAA Technical Memorandum NMFS-NWFSC-59, Extraction, clean-up and Gas Chromatography/Mass Spectrometry analysis of sediments and tissues of organic contaminants, Catherine A. Sloan, Donald W. Brown, Ronald W. Pearce, Richard H. Boyer, Jennie L. Bolton, Douglas G. Burrows, David P. Herman and Margaret M. Krahn, Northwest Fisheries Science Center, Environmental Conservation Division, 2725 Montlake Blvd East, Seattle, Washington 98112, March 2004.

[7] Determination of Polycyclic Aromatic Hydrocarbons (PAHs) in seafood using Gas Chromatography-Mass Spectrometry: A collaborative study, Katerina Mastovski et al. Covance Laboratories Inc. 671 S. Meridian Road, Greenfield, IN 46140.

[8] Evaluation of rapid extraction and analysis techniques for Polycyclic Aromatic Hydrocarbons (PAHs) in seafood by GC/MS/MS, Ed George, Bruker Application Note # GCMS-09 (2015), Bruker Daltonics Inc, Billerica, Massachusetts.

[9] Determination of Polycyclic Aromatic Hydrocarbons in edible seafood by QuEChERS-based extraction and Gas ChromatographyTandem Mass Spectrometry, Yoko S. Johnson, Journal of Food Science 2012; 77(7):131–136.

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Last updated: Mar 15, 2019 at 11:04 AM

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