Evaluation of Rapid Extraction and Analysis Techniques for Polycyclic Aromatic Hydrocarbons (PAHs) in Seafood by GC/MS/MS

GC/MS/MS is the determinative technique used for evaluating the rapid sample preparation methods to analyze Polycyclic Aromatic Hydrocarbons (PAHs) in seafood. QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction followed by cleanup or concentration with stir bar sorptive extraction (SBSE); cleanup with dispersive solid phase extraction (dSPE), and direct analysis using a Chromatoprobe sample introduction device are the three studied preparation techniques. To eliminate matric interference and increase precision and accuracy, the combined use of GC/MS/MS with these sample preparation methods was necessary.

The oil spill in The Gulf of Mexico 2010 created anxiety over the safety concerns of environment and seafood. Hence, laboratories turn to an approved method of analysis. As the existing method was incapable of processing large numbers of samples, organizations began to look for more rapid extraction techniques. The QuEChERS approach, used successfully for the analysis of pesticide residues in a variety of food commodities, seemed to be a logical method to try with seafood as 50-100 samples could be sampled per day with minimal solvent consumption. At present QuEChERS-like extraction method for seafood is being evaluated in an inter-laboratory study. The new method uses gas chromatography-mass spectrometry for detection, allowing either single ion monitoring (SIM) mode or tandem mass spectrometry .

Dispersive solid phase extraction (dSPE) and stir bar sorptive extraction (SBSE) cleanup techniques on QuEChERS seafood extracts are evaluated in this study along with GC/MS/MS as it provided excellent quantitative results in the low to sub-ng/g range.

Experimental

Shrimp, oyster, Atlantic salmon, and blue mussel tissue were some seafood matrices used for the study. Calibration and matrix spikes were prepared and analyzed as described. Three evaluated sample preparation approaches were PAHs by QuEChERS with SBSE – followed by Back Extraction (TBE); with dSPE and express extraction and screening with the Chromatoprobe inlet.

QuEChERS+SBSE

This experiment was on logic that the PAH compounds with minimal co-extraction of matrix material could be absorbed by adding (SBSE), a coating of polydimethylsiloxane on a small magnetic stir bar, to a diluted QuEChERS extract. The extraction process for the seafood samples using this approach is described in the flow chart (Figure 1). Figure 2 describes SBSE with back extraction.

Figure 1. QuEChERS extraction procedure with Back Extraction.

Figure 2. Left figure is the SBSE in diluted seafood extract; Right figure is the device inside a vial containing an insert with 220 uL hexane. Setting the vial on the edge of the stir plate with the magnet turned ON allows the SBSE to be gently agitated during back extraction.

QuEChERS with Dispersive Solid Phase Extraction (dSPE)

Figure 3 describes the QuEChERS extraction procedure. with dSPE.

Figure 3. QuEChERS extraction procedure with dSPE cleanup.

The final experiment was designed to provide a rapid prep-and-screen approach for PAHs. It could be used to gain information on batches of seafood samples. Figures 4 and 5 describe the sample preparation method and Chrompatoprobe technique.

Figure 4. Sample preparation workflow for PAH screening method.

Figure 5. Chromatoprobe inlet. The device is inserted into a programmable injection port to allow for temperature control during analysis. A disposable micro-vial is inserted into the probe tip, which resides inside a standard GC injection liner. The column is typically a short 0.10 mm ID capillary column with a thin coating.

General Instrument Parameters and Consumables

Bruker 300-MS with 450-GC, Combi-PAL Auto sampler Injector Inlets: 1177 Split/splitless, 1079 (in Programmed Temperature mode-PTV), Chromatoprobe accessory for 1079 are used.

Inlet Liners:

• 4 mm Restek Siltek fritted liner for 1177 splitless injections
• 3.4 mm SGE Focus Liner for PTV injections on 1079 Columns: Restek Rxi-5 Sil-MS, 30 M x 0.25 mm x 0.25 μm; DB-1 for use with Chromatoprobe, 2 M x 0.1 mm x 0.1 μm SBSE device for back-extraction experiments, 0.5 mm PDMS x 10 mm length dSPE with Restek Q-sep Q251, 150 mg MgSO4/50mg PSA/50 mg C18, packaged in 2 mL centrifuge tubes

Column and Inlet Conditions:

• Column Oven Program:
  45°C hold 1 min, 200°C @ 10°C/min, hold 0; 270°C @ 5°C/min, hold 0; 300°C @ 10°C, hold 0; 320°C @ 20°C/min, hold 1 min.
• 1177 Splitless mode for 0.9 min, 270°C, 40 psi pulse
• 1079 PTV mode; temp and vent times optimized for hexane (TBE) and Acetonitrile (dSPE)
• Chromatoprobe: 70°C to 350°C @ 200°C/min; Column: 45°C for 1 min; 65°C @ 20°C/min, hold 0; 320°C @ 50/min, hold 1 min

General MS Parameters:

• Source: 300°C
• Collision Gas: Argon, 2 mTorr
• MRM dwell times: 100 ms most transitions with total scan time less than 0.6 min/segment. The s-MRM tool was used to optimize the distribution of MRM segments and sensitivity

Results

QuEChERS + SBSE

Pure acetonitrile solvent was used in preparation of calibration standards. The SBSE device was then back extracted with 220 uL of hexane in a micro vial and injected into the GC/MS/MS. These standards were injected in both standard hot splitless mode and in the PTV mode. Results are presented in Tables 1-5 and Figures 6-8. The response for all the calibration levels especially when PTV is used was excellent. Laboratory background and reagent contamination seen at low ng/g levels was the reason for higher percentage of RSD response for naphthalene. The actual amount of analyte injected on the column for each injection technique is summarized in Table 3. The performance was validated by matrix spikes and standard reference material. An example total ion current (TIC) MRM chromatogram is shown in Figure 8. All spiked seafood was homogenized thoroughly before the QuEChERS extraction.

Figure 6. Example Calibration Curve for Benzo(a)pyrene, 1ng/g to 250 ng/g.

Figure 7. Example PTV calibration injection, MRM 252>250, 0.5 ng/g level for Benzo(b)fluoranthene, Benzo(k)fluoranthene, and Benzo(a) pyrene.

Figure 8. TIC-MRM chromatogram of oyster matrix spike by QuEChERS-SBSE-TBE, 5 ng/g.

Table 1. Calibration statistics for PAHs with SBSE and TBE, 1ng/g to 250 ng/g, 2 μL splitless injection.

Table 2. Calibration statistics for PAHs with SBSE and back-extraction, 0.5 ng/g to 50 ng/g, 8 μL PTV injection.

Table 3. Calibration standard theoretical amounts (assuming 100% absolute recovery) of PAHs injected into the Bruker 300-MS GC/MS/MS system. Based on 3 g seafood with sample preparation described in Figure 1. Sub-ng/g levels are easily detected.

Table 4. 20 ng/g seafood spikes results.

Table 5. ASTM SRM 1974b Blue Mussel tissue, 2 μL splitless injection.

Results Summary

Expensive or complex thermal desorption equipment are not necessary as SBSE can be efficiently back-extracted with a small amount of hexane.

Resulting extract is very clean (no color) and less matrix is better for high-throughput robust methods. (See Figure 9) Better precision and accuracy at 0.1-0.5 ng/g is achieved by the use of 8 μL PTV.C13 labeled internal standards could be used to correct lower recovery of late eluting PAHs observed.

Background naphthalene and other PAHs become magnified laboratory and reagent contamination problems at low concentrations.

Figure 9. Final oyster SBSE extract (left) compared to dSPE extract (right). Less matrix is co-extracted with SBSE.

QuEChERS with Dispersive Solid Phase Extraction (dSPE)

Calibration standards for this method is based upon a 10 g seafood sample; The procedure is described in Figure 2 is used for this method of calibration standards based on a 10 g seafood sample. The standards were prepared in acetonitrile, with the intent to directly inject the final extracts into the GC/MS/MS without performing additional solvent exchange steps. PTV injection is ideal as it allows more sample loading and improves method sensitivity and also avoids peak splitting or tailing of early eluting PAHs. In order to investigate potential contamination and/or recovery loses during the dSPE clean up step, two sets of calibration standards were prepared in acetonitrile. One set, directly injected into the GC/MS/MS and other first treated with Restek Q-sep Q251, 150 mg MgSO4/50mg PSA/50 mg C18, packaged in 2 mL centrifuge tubes. 1 mL of the each standard was added to the 2mL tube, vortexed, and centrifuged, which is the same procedure that a sample extract would follow. Calibration curves (Figure 10) and results are listed in Tables 6-9.

Figure 10. Calibration curves for phenanthrene. Left: Calibration in pure acetonitrile. Right: Calibration with acetonitrile standards treated with Q-Sep Q-251.

Table 6. Calibration statistics using QuEChERs-dSPE method. The table represents standards prepared in pure acetonitrile and injected into the Bruker 300-MS. The standards were not treated with Q-Sep Q251. PTV injection, 6 μL.

Table 7. Calibration statistics using QuEChERs-dSPE method. The standards were treated with Q-Sep Q251. PTV injection, 6 μL.

Table 8. Matrix spikes at 5 ng/g for oyster and shrimp. These values were calculated against untreated acetonitrile calibration standards.

Table 9. SRM 1974b results against untreated acetonitrile calibration standards.

Variable results with high RSDs for the PAHs highlighted in Table 7 were observed with the Q-Sep treated standards. Major variation in analyte responses were observed at levels less than 10 ng/g. The contamination was traced to the 2 mL polypropylene centrifuge tube containing the dSPE reagent. Tests also showed that the contamination could be eliminated or greatly reduced if the reagent is removed from the tube and washed with organic solvents.

Shrimp and oyster seafood spikes, along with the ASTM 1974b SRM material, were analyzed to evaluate method performance. As expected, high biased results were observed due to contamination of the dSPE reagent. Results were better against the dSPE treated calibration standards, though it is not recommended since reagent contamination cannot be reasonably controlled.

Results Summary

Convenient packaging of dSPE materials in 2mL centrifuge tubes bodes well for high production labs.

The QuEChERS-dSPE method with PTV injection is quicker and easier than solvent exchanges/traditional silica-gel type clean-ups.

Good recovery of all PAHs was obtained using the technique.

Contamination seen at low ng/g levels originated from dSPE reagent packaging ia a main problem.

For low-level work, removal of the reagents from the packaging and clean with organic solvents is recommended.

QuEChERS “Express” Extraction and Screening with the Chromatoprobe Inlet

A semi-quantitative screening method with Chromatoprobe provided reliable data for levels above 20 ng/g. Seafood samples were rapidly extracted with ethyl acetate, followed by centrifugation. Figure 11 shows a 100 ng/g standard run in under 6 minutes, with relatively good separation and response. Figure 12 shows the ASTM SRM 1974b with Chromatoprobe.

Figure 11. 100 ng/g standard with Chromatoprobe, TIC MRM chromatogram.

Figure 12. ASTM SRM 1974b with Chromatoprobe.

Results Summary

It is ideal for screening, especially for seafood above 20 ng/g as the method is rapid. Carryover is reduced by heating injector to 350C at the end the GC cycle and limiting amount of extract to 1-2 μL added to micro- vial. Limited to manual injections only.

Conclusion

Good precision and accuracy for subng/g detection limits is provided alsong with effective removal of matrix interference when the Bruker 300-MS triple quadrupole mass spectrometer is used. The advantages of cleaner extracts and analyte enrichment via back extraction with a small volume of GC-suitable solvent (hexane) are demonstrated in the QuEChERS-SBSE-BE method. However, late-eluting PAHs was observed. The QuEChERS-dSPE cleanup method using commercially prepared dSPE reagents provided excellent recovery for all PAHs studied. Contamination was observed in calibration standards processed with the reagents, and was traced to the packaging. PTV is particularly important for extracts prepared in acetonitrile due to potential peak splitting for early eluting PAHs. The Chromatoprobe device provided a good screening tool for PAHs in seafood. Levels greater than or equal to 20 ng/g in seafood were easily detected. Careful attention to potential carryover from highly contaminated samples is required.

Shrimp and oyster seafood spikes, along with the ASTM 1974b SRM material, were analyzed to evaluate method performance. High biased results were observed due to contamination of the dSPE reagent. Results were better against the dSPE-treated calibration standards, though not recommended since reagent contamination cannot be reasonably controlled.

This information has been sourced, reviewed and adapted from materials provided by Bruker Life Sciences Mass Spectrometry.

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