is an example chromatogram generated from salmon spike-recovery experiments demonstrating sufficient separation/detection of 33 PAHs and substituted PAHs by GC-MS in 36 minutes at a sample overspike concentration of 500 ng/g (wet weight). summarizes native PAH and deuterated internal standard GC-MS instrumental parameters and detection limits. Benz[a]anthracene was the only compound that had a coefficient of determination (r2) less than 0.99 (r2 = 0.983); all others had coefficients ≥ 0.99 within the calibration range of 1 – 1000 pg/μL, demonstrating excellent method linearity. Parent and substituted PAH instrumental detection limits were assigned to PAH molecular ions when their lowest abundance confirmation ion signal-to-noise (S/N) ≥ 3 as determined by the signal-to-noise script of the Agilent MSD ChemStation data analysis software, version E (Santa Clara, CA). Samples used in the determination of instrumental detection limits were standard solutions analyzed from several batches over several days. The instrumental detection limits for quantified analytes ranged from 1 to 5 pg/μL. Analytes were considered quantitative when they calibrated with r2 ≥ 0.98, their lowest abundance confirmation ion had S/N > 3 and had reproducible and accurate quantitation (± 20% of their true value) as assessed from continuing calibration verification standards. All parent and substituted PAHs met these criteria. Sample residues that met all criteria but had S/N < 3 were designated below detection limit (BDL), while those that did not meet one or more of the above criteria were designated non-detectable (ND).
Representative selective ion monitoring total ion current (TIC) for PAHs in smoked salmon with a 500 ng/g wet weight overspike. PAHs corresponding to chromatogram numbers can be found in .
summarizes PAH spike recoveries obtained for traditional acetonitrile based QuEChERS extraction methods E1 and E2 from smoked salmon with 3, 8, and 11% fat content. Recoveries from smoked salmon using extraction scheme E1 (1% acetic acid in acetonitrile and AOAC salts) yielded low recoveries, on average less than 67%, with individual PAH recoveries typically ranging from 35 to 87%. Extraction scheme E2 (acetonitrile and EN salts) performed equally poorly, with average PAH recoveries being less than 68% and individual PAH recoveries ranging from 24 to 88%. Both extraction scheme E1 and E2 were especially poor at recovering 2-, 3-, 5- and 6-ring PAHs, where average recoveries across this subgroup of PAHs were 57% and 56% respectively.
Traditional QuEChERS method performance in recovering 33 PAHs (mean ± RSD; n = 4) from increasing fat content smoked salmon fortified at 500 ng/g wet weight.
Variant QuEChERS solvent systems have been described for the analysis of pesticide residues in fruits (14
). Additionally, the individual and combined performance of various ratios of ethyl acetate, acetone, hexane, methylene chloride, acetonitrile, cyclohexane and iso-octane have been reported for multiple residue pesticide methods and EPA methods for extraction of nonvolatile and semivolatile organic compounds from solid and semi-sold samples (10
). Of interest were solvent systems with improved selectivity for non-polar PAH residues and that were lower in cost than acetonitrile. It was found that a three-component variant solvent system of acetone, ethyl acetate and iso-octane (2:2:1; v/v/v) met these criteria.
summarizes PAH recoveries obtained from smoked salmon using modified QuEChERS extraction schemes E3 and E4. Extraction scheme E3 (acetone, ethyl acetate, iso-octane and AOAC salts) led to good recoveries, on average 90% over all fish tested. Notable performance gains were made for 2-, 3- and 5-ring PAHs where recoveries were improved 50–200%, while recoveries of 4- and 6-ring PAHs were slightly improved by ~ 30–45% as compared to acetonitrile. Extraction scheme E4 (acetone, ethyl acetate, iso-octane and EN salts) performed equally well, with an average PAH recovery of 87% across all fish and individual PAHs displaying the same range of improvement as extraction scheme E3. Additionally, both extraction scheme E3 and E4 displayed good extraction precision with relative standard deviations typically less than 10% for all fish tested.
Modified QuEChERS method performance in recovering 33 PAHs (mean ± RSD; n = 4) from increasing fat content smoked salmon fortified at 500 ng/g wet weight.
It is well understood that the planar hydrophobic chemical structure of PAHs leads to their association with fatty components of biological matrices (i.e. waxes, lipids, steroids and pigments). Extraction conditions that disrupt these associations should give rise to enhanced extraction performance. A solvent’s ability to disrupt interactions may be assessed by comparison of solvent and PAH octanol-water partition coefficients (log KOW
), where solvents with coefficients similar to PAHs should display enhanced selectivity. The log KOW
for PAHs used in this study ranged from 3.3 for naphthalene to 7.7 for dibenzo[a,l
]pyrene. Acetonitrile has a reported log KOW
= −0.34, while values for acetone, ethyl acetate and iso-octane are −0.24, 0.73 and 4.1 respectively. It has also been demonstrated that extraction of various food stuffs with acetonitrile resulted in limited extraction of hydrophobic matrix components (14
). This information coupled to our results suggests that the three component extraction solvent used in extraction schemes E3 and E4 possesses physicochemical characteristics that allow it to interact more intimately with fatty fish matrices. At the molecular level, extraction with acetone, ethyl acetate and iso-octane may lead to improved recoveries by allowing water miscible acetone and ethyl acetate to recover PAHs trapped in water-sealed matrix pores and making them available for transfer to iso-octane.
It is also known that increased extraction temperatures can disrupt analyte-matrix interactions by decreasing the activation energy required for analyte desorption processes and decreasing solvent viscosity, facilitating better solvent-matrix penetration (12
). It was found that addition of magnesium sulfate containing extraction salts generated sample extraction temperatures of 45 – 50°C that persisted for the duration of the extraction/partition procedure (data not shown). Extraction temperatures in the range observed have been by reported by others and should increase solvent capacity for PAHs (12
). The findings presented indicate that the improved extraction performance of schemes E3 and E4 likely resulted from the combined influence of enhanced solvent selectivity and elevated sample extraction temperatures.
In order to evaluate the effectiveness and utility of the modified QuEChERS methods developed in this study, a comparison to other published extraction techniques is presented () (13
). Compared to soxhIet extraction with hexane, it was found that modified QuEChERS methods substantially improved average recovery of all 15 PAHs by roughly 38% and led to individual gains of 50–125% for naphthalene, anthracene, benz[a
]fluoranthene and dibenz[a,h
]anthracene. Similar overall improvements were demonstrated when compared to accelerated solvent extraction (ASE) with hexane. Interestingly, the performance of modified QuEChERS methods was comparable to a validated dichloromethane and acetonitrile (1:1; v/v) based ASE extraction method in average recovery across all PAHs. However, modified QuEChERS methods showed improved recoveries in lower molecular weight PAHs, while ASE performed better at recovering benzo[g,h,i
]perylene. Estimated method detection limits (MDL) for modified QuEChERS methods are presented in in relation to FDA PAH levels of concern in shrimp, crab, oysters and finfish (25
). MDLs were defined as the product of the analyte instrument detection and the method dilution factor, which was a factor of 2 in this case. MDLs were all well below levels of concern in these food stuffs, demonstrating the potential utility of the developed methods. If needed, additional method sensitivity could be achieved through the introduction of a solvent reduction procedure prior to instrumental analysis.
Modified QuEChERS extraction performance (% recovery ± SD) compared to literature reported soxhlet and accelerated solvent extraction (ASE) in recovering 15 PAHs from fish tissues.
Comparison of FDA PAH levels of concern in relation to estimated modified QuEChERS method detection limits (MDL).
A comparison of PAH levels measured in commercially available smoked salmon using both modified QuEChERS extraction schemes E3 and E4 is presented in . Results for the two methods were quite similar across all fish in terms of PAH profile, quantitation, extraction precision and sum of quantified PAHs (ΣPAHs). Naphthalene, 2-methylnaphthalene, 1-methylnaphthalene, phenanthrene, fluoranthene, pyrene and benzo[g,h,i
]perylene were consistently detected. Anthracene was observed only in 3% fat salmon, but was not quantified because it did not meet signal-to-noise limits. Levels of individual PAHs typically fell in a range of 5–60 ng/g wet weight with fluoranthene and benzo[g,h,i
]perylene displaying the lowest levels and naphthalene, 2-methylnaphthalene and pyrene consistently accounting for > 70% of total PAH mass recovered. Extraction schemes E3 and E4 also performed equally well with regards to extraction precision (RSD < 20%) and produced nearly identical ΣPAH values within and across the fat levels used. Results from this study are comparable to other studies in terms of PAH profile, range and summed residue loads and, together with spike-recovery experiments, demonstrate that choice of extraction solvent is crucial to extraction performance (6
Comparison of PAH levels (mean ng/g wet weight ± SD, n = 3) measured in commercially available smoked salmon by two new modified QuEChERS extraction schemes
The goal of the current study was to develop and validate a multi residue method for the analysis of PAHs and their substituted derivatives in high-fat smoked salmon. The data presented strongly indicate that a QuEChERS based analytical platform implementing a three-component acetone, ethyl acetate and iso-octane extraction solvent in a 2:2:1 (v/v/v) ratio coupled to dispersive SPE sample cleanup and GC-MS is a fast, selective, efficient and precise method for the determination of PAHs in high-fat smoked fish products. The modified QuEChERS methods described show good potential for use in monitoring levels of PAHs in lipid-rich fish.