Determination of PAH in Seafood: Optimized Sample Preparation Procedures For LC-Fluorescence Screening and GC-MS(MS) Confirmation

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1 Determination of PAH in Seafood: Optimized Sample Preparation Procedures For LC-Fluorescence Screening and GC-MS(MS) Confirmation Michael S. Young, Mark E. Benvenuti, Jennifer A. Burgess and Kenneth J. Fountain Waters Corporation, Milford, MA USA APPLICATION BENEFITS Rapid extraction of seafood using proven QuEChERS methodology LC-Fluorescence analysis in under five minutes with no further sample preparation required Straight-forward SPE cleanup for GC-MS(MS) confirmation Waters solutions DisQuE Dispersive Sample Preparation Products for QuEChERS INTRODUCTION PAH (polycyclic aromatic hydrocarbons) are toxic compounds common in nature and are constituents of coal and petroleum. Many of these compounds (for example, benzo(a)pyrene) are carcinogenic. The recent major oil spill in the Gulf of Mexico has focused attention on the problem of PAH contamination and also on the challenges related to PAH analysis of food and environmental samples. In this presentation, we will discuss an optimized sample preparation protocol for determination of PAH in shellfish samples. The initial sample extraction is performed using the QuEChERS dispersive method (Quick, Easy, Cheap, Effective, Rugged, and Safe) by which a sample is extracted with acetonitrile in the presence of an excess of buffer salts. This technique provides a convenient extract well suited for LC analysis with fluorescence detection (LC-FL); for the LC-FL analysis, no further workup is required (a detailed presentation of the LC-FL procedure is presented in reference 1). A similar approach has been successfully utilized to rapidly screen a large number of seafood samples impacted by the Gulf of Mexico oil spill. 2 Any PAH compounds detected by the LC-FL method above concern levels may require GC-MS confirmation. The same QuEChERS extract used for LC-FL screening can be used for GC-MS confirmation. However, for optimum GC-MS performance the extract should be cleaned up and exchanged to a more suitable GC solvent. A simple, straightforward solid-phase extraction (SPE) strategy is presented for effective PAH confirmation analysis by GC-MS(MS) in oyster and related samples prepared using the QuEChERS approach. ACQUITY H-Class System with Fluorescence Detection Quattro micro GC Mass Spectrometer Certified Sep-Pak Silica Cartridge Oasis HLB Cartridge key words Polycyclic Aromatic Hydrocarbons (PAH), GC-MS(MS), SPE, Shellfish 1

2 EXPERIMENTAL GC Conditions System: Agilent 6890 Column: Rxi -5Sil, 30 m x 0.25 mm (i.d.), 0.25 µm (df) Injection Volume: 1.0 µl Injection Mode: Splitless (purge time 0.75 min) Carrier Gas: Helium Flow Rate: 0.8 ml/min (constant flow) Temp. Program: 50 o C initial, hold 1 min, then 10 o C/min to 310 o C, hold 10 min MS Conditions System: Waters Quattro micro GC Ion Mode: EI+ Ion Energy: 70 ev Inter Channel Delay: 0.01 sec Dwell: 0.03 sec Table 1 summarizes the collision energies and MRM transitions used for this study. These values were adapted from reference 3. PAH MRM1 Collision (ev) MRM2 Collision (ev) Function 1 1. Naphthalene 128> > Acenaphthylene 154> > Acenaphthene 152> > Fluorene 166> > ISTD1: Acenaphthene-d10 162> Function 2 5. Phenanthrene 178> > Anthracene 178> > Fluoranthene 202> > Pyrene 202> > Benz(a)anthracene 228> > Chrysene 228> > ISTD2: Chrysene-d12 240> Function Benzo[b]fluoranthene 252> > Benzo[k]fluoranthene 252> > Benzo[a]pyrene 252> > Indeno(1,2,3-cd)pyrene 276> > Dibenz(a,h)anthracene 278> > Benzo[ghi]perylene 276> > ISTD3: Perylene-d12 264> Table 1. MRM transitions and collision energies. 2 Determination of PAH in Seafood: Optimized Sample Preparation Procedures for LC-Fluorescence Screening and GC-MS(MS) Confirmation

3 Sample Preparation Step 1: Initial Extraction (QuEChERS) Place 15 g homogenized sample into 50 ml centrifuge tube. Add contents of DisQuE tube (p/n ) and 15 ml acetonitrile (ACN). Shake the tube for 1 minute and then centrifuge for rpm. For LC analysis, transfer a suitable portion of top (ACN) layer to LC vial. For GC-MS analysis, transfer a 1.0 ml aliquot of the top layer to a suitable vial or test tube to prepare for SPE cleanup. Step 2: SPE Cleanup To the 1 ml aliquot of supernatant (from Step 1), add internal standards, mix well and then add 2 ml water. Proceed to SPE cleanup using an Oasis HLB cartridge followed by Certified Sep-Pak Silica cartridge. (See SPE details in Figure 1) SPE with the Oasis HLB cartridge accomplishes solvent exchange to hexane with no loss of volatile constituents. SPE with Certified Sep-Pak Silica removes non-polar interferences (by hexane wash), removes polar interferences (by retention) and concentrates PAH into eluent optimal for GC injection. Sample Pre-preparation Take 1 ml of the top (ACN) layer from the DisQuE Extraction, add internal standards and dilute to 3 ml with water. CARTRIDGE 1 CARTRIDGE 2 Oasis HLB Cartridge 3 cc, 60 mg Certified Sep-Pak Silica Cartridge 3 cc, 500 mg Sample Pre-preparation 1 ml acetonitrile (ACN), 1 ml 25:75 ACN/water Condition 2 ml hexane Attach Cartridge 1 to Cartridge 2 with adaptor Load diluted extract Wash 1 ml 50:50 ACN/water and dry cartridge under vacuum for a few minutes Go to conditioned Sep-Pak Silica Cartridge 2 Wash 2 ml hexane (discard) Install collection vessels Elute 3 ml 25:75 DCM/Hexane Evaporate to 0.25 ml (not to dryness!) Figure 1. SPE Cleanup Protocols, Oasis HLB (left), Certified Sep-Pak Silica (right) Determination of PAH in Seafood: Optimized Sample Preparation Procedures for LC-Fluorescence Screening and GC-MS(MS) Confirmation 3

4 RESULTS Figure 2 shows a reconstructed GC-MS(MS) chromatogram obtained from analysis of an oyster sample spiked with 16 priority PAH at a 35 ng/g level. Shrimp analysis was similar. Figure 3 shows the extracted ion chromatograms for benzo(a)pyrene obtained from an oyster sample spiked at the 5 ng/g level ,12 3: MRM of 7 Channels EI+ TIC 9.77e % , min Figure 2. GC-MS(MS) reconstructed TIC chromatograms of oyster sample spiked at 35 ng/g (compound ID as presented in Figure 1, internal standards were omitted for clarity) : MRM of 7 Channels EI+ 252 > e3 0% : MRM of 7 Channels EI+ 252 > e3 0% min Figure 3. GC-MS(MS) chromatograms obtained from oyster spiked at 5 μg/kg for two MRM transitions (blue trace is for spiked sample, red trace is from blank oyster). 4 Determination of PAH in Seafood: Optimized Sample Preparation Procedures for LC-Fluorescence Screening and GC-MS(MS) Confirmation

5 Table 2. SPE recovery data (n=3). PAH % Recovery (% RSD) 1. Naphthalene 90 (8.0) 2. Acenaphthylene 92 (3.2) 3. Acenaphthene 99 (6.7) 4. Fluorene 95 ( Phenanthrene 85 (9.1) 6. Anthracene 94 (6.1) 7. Fluoranthene 94 (4.2) 8. Pyrene 97 (4.7) 9. Benz(a)anthracene 97 (6.6) 10. Chrysene 86 (6.6) 11: Benzo[b]flouranthene 92 (2.1) 12: Benzo[k]flouranthene 90 (5.5) 13. Benzo[a]pyrene 90 (1.9) 14. Indeno(1,2,3-cd)pyrene 90 (8.6) 15. Dibenz(a,h)anthracene 93 (5.2) 16. Benzo[ghi]pertlene 106 (4.8) Using internal standard calculation, correlation (r 2 ) was or better for all PAH (5 point matrix matched curve range 5 to 100 ng/g) in oyster or shrimp. SPE recovery was measured from results obtained from 3 replicates prepared in oyster matrix; the recoveries ranged from 85 to 106% and RSD ranged from 2 to 9% (see Table 2). The recovery experiment was performed by comparison of response for samples spiked prior to SPE compared with response for samples spiked after SPE. Also, for all recovery samples, the internal standards were spiked after SPE. DISCUSSION The QuEChERS extraction procedure has been shown to be effective for the extraction of PAH compounds from seafood prior to LC analysis with fluorescence detection. 1,2,4 This LC analysis provides a powerful screening procedure with detection limits below 10 ng/g. However, compounds identified using LC-FL may require confirmation by a second technique. GC-MS is commonly used for PAH analysis; GC-MS(MS) allows for greater sensitivity and selectivity for PAH confirmation analysis. Although the QuEChERS extract is suitable for LC analysis with no further cleanup, solvent exchange and sample cleanup is recommended prior to GC-MS(MS). In the effective SPE cleanup process presented here, the acetonitrile extract is exchanged to hexane, a much more suitable GC solvent. Also, potential interferences such as fats, aliphatic hydrocarbons, polar compounds and pigments are removed from the extract. Cleaner chromatograms and a cleaner GC instrument are the results. Determination of PAH in Seafood: Optimized Sample Preparation Procedures for LC-Fluorescence Screening and GC-MS(MS) Confirmation 5

6 CONCLUSIONS The dispersive sample preparation (QuEChERS) used for LC-FL provides an extract that can be readily utilized for GC-MS(MS) confirmation. A straightforward SPE protocol is demonstrated for sample cleanup and solvent exchange to provide optimum GC performance. The SPE and GC-MS(MS) approach provides effective confirmation analysis. REFERENCES 1. Waters Application Note EN, Ensuring Seafood Safety With Rapid Screening for Polyaromatic Hydrocarbons Using LC-Fluorescence, Ann M. Thayer, Testing Gulf Seafood, Chem. & Eng. News, 2011, 89, Waters Application Note EN, Fast GC/MS/MS Analysis of Polyaromatic Hydrocarbons (PAHs) using the Waters Quattro micro GC, Maria Joao Ramalhosa et. al., Analysis of Polycyclic Aromatic Hydrocarbons in Fish: Evaluation of a Quick, Easy, Cheap, Effective, Rugged, and Safe Extraction Method, J. Sep. Sci., 2009, 32, For GC-MS analysis, the QuEChERS acetonitrile extraction protocol provides equivalent performance compared with ethyl acetate or methylene chloride extraction of seafood. Waters, The Science of What s Possible, DisQuE, ACQUITY, Quattro micro GC, Sep-Pak, and Oasis are trademarks of Waters Corporation. Rxi is a registered trademark of Restek Corporation. All other trademarks are the property of their respective owners Waters Corporation. Produced in the U.S.A. September EN LS-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: