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
A P P LI C ATION B ENE F ITS ■■
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
Wat e r s s o lu t i o n s DisQuE ™ Dispersive Sample Preparation Products for QuEChERS
INTRODU C TION 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
E X P ERIM ENTAL
Table 1 summarizes the collision energies and MRM transitions used for this study. These values were adapted from reference 3.
GC Conditions System:
Agilent 6890
Column:
Rxi®-5Sil, 30 m x 0.25 mm (i.d.), 0.25 µm (df)
Function 1
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
PAH
MRM1
Collision (eV)
MRM2
Collision (eV)
1. Naphthalene
128>102
20
128>128
15
2. Acenaphthylene
154>153
20
154>152
30
3. Acenaphthene
152>151
20
152>150
25
4. Fluorene
166>165 162>160
20
166>164
35
5. Phenanthrene
178>152
15
178>151
40
6. Anthracene
178>152
15
178>151
40
7. Fluoranthene
202>200
35
202>202
20
8. Pyrene
202>200
35
202>202
20
9. Benz(a)anthracene
228>226
30
228>228
25
10. Chrysene
228>226
30
228>228
25
ISTD2: Chrysene-d12
240>236
35
11. Benzo[b]fluoranthene
252>250
30
252>252
25
ISTD1: Acenaphthene-d10
20
Function 2
Function 3
System:
Waters Quattro micro GC
Ion Mode:
EI+
12. Benzo[k]fluoranthene
252>250
30
252>252
25
Ion Energy:
70 eV
13. Benzo[a]pyrene
252>250
30
252>252
25
Inter Channel Delay:
0.01 sec
14. Indeno(1,2,3-cd)pyrene 276>274
40
276>276
25
Dwell:
0.03 sec
15. Dibenz(a,h)anthracene
278>276
35
278>278
25
16. Benzo[ghi]perylene
276>274
40
276>276
25
ISTD3: Perylene-d12
264>260
30
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
Sa mp l e P r e pa r at i o n Step 1: Initial Extraction (QuEChERS) Place 15 g homogenized sample into 50 mL centrifuge tube. Add contents of DisQuE tube (p/n 186004571) and 15 mL acetonitrile (ACN). Shake the tube for 1 minute and then centrifuge for 5 minutes @ 3000 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
Sep-Pak Silica Cartridge
Sample Pre-preparation
Condition
3 cc, 60 mg
Certified
3 cc, 500 mg
1 mL acetonitrile (ACN), 1 mL 25:75 ACN/water
2 mL hexane Attach Cartridge 1 to Cartridge 2 with adaptor
Load diluted extract
Wash 2 mL hexane (discard) Install collection vessels
Wash
Elute
1 mL 50:50 ACN/water and dry cartridge under vacuum for a few minutes
3 mL 25:75 DCM/Hexane
Go to conditioned Sep-Pak Silica Cartridge 2
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
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. 11,12
100
8 1
3
3: MRM of 7 Channels EI+ TIC 9.77e3
4 7
13 10
2
9
14,15
%
5
6 16
0 10
12
14
16
18
20
22
24
26
28
30 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). 100
%
3: MRM of 7 Channels EI+ 252 > 250 1.26e3
0 27.2
27.4
27.6
27.8
28.0
100
28.2
%
3: MRM of 7 Channels EI+ 252 > 252 1.03e3
0 27.2
27.4
27.6
27.8
28.0
28.2 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
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 (5.7
5. 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 (r2) was 0.995 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.
DIS C USSION 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
REFERENCES
C ON C LUSIONS ■■
■■
The dispersive sample preparation (QuEChERS) used for LC-FL provides an extract that can be readily utilized for GC-MS(MS) confirmation.
1. Waters Application Note 720003891EN, “Ensuring Seafood Safety With Rapid Screening for Polyaromatic Hydrocarbons Using LC-Fluorescence,” 2011
A straightforward SPE protocol is demonstrated for sample cleanup and solvent exchange to provide optimum GC performance.
3. Waters Application Note 720001910EN, “Fast GC/MS/MS Analysis of Polyaromatic Hydrocarbons (PAHs) using the Waters Quattro micro GC,” 2007
■■
The SPE and GC-MS(MS) approach provides effective confirmation analysis.
■■
For GC-MS analysis, the QuEChERS acetonitrile extraction protocol provides equivalent performance compared with ethyl acetate or methylene chloride extraction of seafood.
2. Ann M. Thayer, “Testing Gulf Seafood”, Chem. & Eng. News, 2011, 89, 12-16
4. 4. 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, 3529–3538
Waters, T he Science of W hat’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. ©2011 Waters Corporation. Produced in the U.S.A. September 2011 720004162EN LS-PDF
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
