Jackie Whitecavage, Jack R. Stuff und Edward A. Pfannkoch,
Jeffrey H. Moran,
Extract of a shrimp sample (3 g), spiked with a mixture (2,5 ppb) of naphthalene (1), fluorene (2), phenanthrene (3), anthracene (4), fluoranthene (5), pyrene (6), benzo[a]anthracene (7), chrysene (8) and benzo[a]pyrene (9).
Extract of a oyster sample (3 g), spiked with a mixture (2,5 ppb) of naphthalene (1), fluorene (2), phenanthrene (3), anthracene (4), fluoranthene (5), pyrene (6), benzo[a]anthracene (7), chrysene (8) and benzo[a]pyrene (9).
Extract of a fish sample (3 g), spiked with a mixture (2,5 ppb) of naphthalene (1), fluorene (2), phenanthrene (3), anthracene (4), fluoranthene (5), pyrene (6), benzo[a]anthracene (7), chrysene (8) and benzo[a]pyrene (9).
Calibration curve for Benzo[a]pyrene determined in spiked oysters. d12-perylene was added as internal standard (IS) to the sample resulting in a concentration of 25 ppb IS in the sample. Further, the following deuterated internal standards were used: for the analytes naphthalene and fluorene: d8-naphthalene; for anthracene and phenanthrene: d10-phenanthrene; and for fluoranthene, pyrene, benz[a]anthracene and chrysene: d12- chrysene.
Polyaromatic seafood platter?
The oil well disaster in the Gulf of Mexico could have wide-ranging consequences for the environment and potentially for the tens of thousands who make a living from delivering seafood to our plates. Closer to home, consumers are still pondering whether it is safe to put seafood on the menu. A simple answer is not easy to come by, but government and industry resources at many levels have been actively focusing on the issue. Analytical labs have been working overtime using cumbersome regulated methods while trying to find new and more productive ways to analyze the mountain of samples before them. As always, when we rise to a challenge, new and sometimes unexpected answers are found.
This much seems certain, nobody can really tell how the marine biosphere will respond to the trauma - or to the treatment for that matter. Following the unprecedented oil spill in the Gulf of Mexico that lasted almost three months, the environment has had to contend with the two-pronged attack of crude oil and of chemicals added to the cocktail to disperse the black gold. But which traces will remain? - And will toxic compounds accumulate? - And where? These are among the questions being posed. And, as always, the main focus of attention is potential direct health effects to humans. The main interaction between man and the environment of the Gulf of Mexico is through the plentiful supply of seafood harvested there. There has been general agreement that pollutants could wind up in our food and, notably, that polyaromatic hydrocarbons (PAHs) could accumulate through the food chain and be served up in concentrated form on a seafood platter. If so, what could appropriate measures to counter the threat look like?
The answer and first step has been to implement comprehensive controls. As it turns out, every answer gives rise to new questions: The official method used to determine PAH levels in seafood is NOAA NMFS-NWFSC-59, which relies on Accelerated Solvent Extraction (ASE), two separate evaporative concentration steps, liquid chromatography cleanup, and finally GC/MS analysis. Following this method means that typically only one batch of 14 samples and associated standards can be analyzed per week in most laboratories. When faced with a mountain of samples, initial estimates ran as high as 10,000 samples per month, more efficient methods will have to be found.
Extraction technique of choice
The authors of a recent publication set out to find a more efficient and practicable quantitative analysis method for PAHs in seafood, performing a study to determine if using a QuEChERS (Quick, Easy, Cheap, Effected, Rugged, and Safe) extraction method in conjunction with Stir Bar Sorptive Extraction (SBSE) could meet regulatory limits of detection and requirements established for precision and accuracy. SBSE has proven its worth for many challenging matrices over the past decade. A recent EPA Region 7 study has shown that SBSE is an effective and fast technique for trace PAH determination in water. The results of the study were presented in May 2010 at the 58th American Society for Mass Spectrometry (ASMS) Conference.
SBSE relies on the GERSTEL Twister™, a glass coated magnetic stir bar with an external layer of polydimethylsiloxane (PDMS). While stirring the sample, the Twister efficiently extracts organic compounds into the PDMS phase. Following the extraction step, the Twister is removed from the sample, quickly dried with a lint-free cloth and placed in a thermal desorption tube. The tube and twister are then placed in an autosampler tray, in which the tube is kept sealed to eliminate the risk of contamination. Thermal desorption of one or more Twisters is performed, for example, in the GERSTEL Thermal Desorption System (TDS) or Thermal Desorption Unit (TDU) and the analytes are transferred directly and quantitatively to the GC/MS system.
Compared with the NOAA method mentioned earlier, the QuEChERS-SBSE- GC/MS method is a revolution, no less; the QuEChERS method uses a single-step acetonitrile (ACN) extraction and liquid–liquid partitioning based on salting out from the water in the sample using MgSO4. The original QuEChERS procedure for pesticides includes dispersive-solid-phase extraction (dSPE) cleanup to remove organic acids, excess water, and other components with a combination of primary secondary amine (PSA) sorbent and MgSO4. However, this cleanup step provides no additional concentration factor making it difficult to achieve detection limits meeting the current requirements for PAH analysis.
The procedure used in the work reported here includes using SBSE as a combined cleanup and concentration step, eliminating organic acids and other polar and high molecular weight matrix components and providing a substantial concentration factor to easily achieve the regulatory detection limits. In brief, 3 g of a homogenized seafood tissue sample in water is extracted with ACN in a 50 mL centrifuge tube followed by addition of 6.0 g MgSO4 and 1.5 g sodium acetate which is shaken and centrifuged. A portion of the ACN extract (upper layer) is added to a 10 mL vial along with 4 mL 0.1 M NaHCO3 and a GERSTEL Twister™ stir bar that is used to extract and concentrate the PAHs. The Twister is removed from the sample extract, rinsed with DI water to remove matrix residue, dried with a lint-free cloth, placed in a TDU tube, and the TDU tube is placed in the MultiPurpose Sampler (MPS) tray. From that point on, everything is automated. The Twister is thermally desorbed and analytes are transferred to the Cooled Injection System (CIS) GC inlet where they are cryofocused. Using a fast temperature program, the focused analytes are transferred in a narrow band from the CIS inlet to the GC column, providing the best possible basis for a clean GC separation and ultra-low limits of detection. The system used combined a GERSTEL MPS, TDU and CIS 4 with a GC/MS System from Agilent Technologies (GC 7890/MSD 5975). Using this method, at least 40 samples can be analyzed per day.
The scientists showed that QuEChERS and SBSE extraction is an excellent alternative to the currently used NOAA NMFS-NWFSC-59 method used for the determination of PAHs in seafood matrices. SBSE was able to outperform the NOAA method on multiple counts: 1) Efficient, easy, and conveniently automated, SBSE-GC/MS dramatically improves sample throughput, which is especially important when a large number of samples need to be analyzed or screened. 2) Even though the NOAA method relies on two evaporative analyte concentration steps and LC clean-up, QuEChERS-SBSE-GC/MS provides limits of detection that are a factor 10 – 50 lower. Reducing the 1:10 split ratio used in the SBSE-GC/MS method would further improve detection limits. 3) The QuEChERS-SBSE-based method significantly reduces the amount of solvent needed. This saves cost, protects the environment, and reduces the amount of solvent in the laboratory air thereby improving occupational safety for laboratory staff.
|AppNote-2010-06 part a|
Alternative Procedure for Extraction and Analysis of PAHs in Seafood by QuEChERS-SBSE-GC-MS
|AppNote-2010-06 part b|
High Throughput Method for the Determination of PAHs in Seafood by QuEChERS-SBSE-GC-MS