Determination of Polycyclic Aromatic Hydrocarbons (PAHs) in Seafood Using Gas Chromatography-Mass Spectrometry: Collaborative Study
Authors: Mastovska Katerina , Sorenson Wendy R , Hajslova Jana ,   Collaborators:, Betzand J , Binkley J , Bousova K , Cook J M , Drabova L , Hammack W , Jabusch J , Keide K , Lizak R , Lopez-Sanchez P , Misunis M , Mittendorf K , Perez R , Perez S , Pugh S , Pulkrabova J , Rosmus J , Schmitz J , Staples D , Stepp J , Taffe B , Wang J , Wenzl T 
Published: April 2015

Abstract
A collaborative study was conducted to determine selected polycyclic aromatic hydrocarbons (PAHs) and their relevant alkyl homologs in seafood matrixes using a fast sample preparation method followed by analysis with GC/MS. The sample preparation method involves addition of 13C-PAH surrogate mixture to homogenized samples and extraction by shaking with a water–ethyl acetate mixture. After phase separation induced by addition of anhydrous magnesium sulfate–sodium chloride (2 + 1, w/w) and centrifugation, an aliquot of the ethyl acetate layer is evaporated, reconstituted in hexane, and cleaned up using silica gel SPE. The analytes are eluted with hexane–dichloromethane (3 + 1, v/v), the clean extract is carefully evaporated, reconstituted in isooctane, and analyzed by GC/MS. To allow for the use of various GC/MS instruments, GC columns, silica SPE cartridges, and evaporation techniques and equipment, performance-based criteria were developed and implemented in the qualification phase of the collaborative study. These criteria helped laboratories optimize their GC/MS, SPE cleanup, and evaporation conditions; check and eliminate potential PAH contamination in their reagent blanks; and become familiar with the method procedure. Ten laboratories from five countries qualified and completed the collaborative study, which was conducted on three seafood matrixes (mussel, oyster, and shrimp) fortified with 19 selected PAH analytes at five different levels of benzo[a]pyrene (BaP) ranging from 2 to 50 μg/kg. Each matrix had a varying mixture of three different BaP levels. The other studied PAHs were at varying levels from 2 to 250 μg/kg to mimic typical PAH patterns. The fortified analytes in three matrixes were analyzed as blind duplicates at each level of BaP and corresponding other PAH levels. In addition, a blank with no added PAHs for each matrix was analyzed singly. Eight to 10 valid results were obtained for the majority of determinations. Mean recoveries of all tested analytes at the five different concentration levels were all in the range of 70–120%: 83.8–115% in shrimp, 77.3–107% in mussel, and 71.6–94.6% in oyster, except for a slightly lower mean recovery of 68.6% for benzo[ a ]anthracene fortified at 25 μg/kg in oyster (RSDr: 5.84%, RSDR: 21.1%) and lower mean recoveries for anthracene (Ant) and BaP in oyster at all three fortification levels (50.3–56.5% and 48.2–49.7%, respectively). The lower mean recoveries of Ant and BaP were linked to degradation of these analytes in oyster samples stored at –20°C, which also resulted in lower reproducibility (RSDR values in the range of 44.5–64.7% for Ant and 40.6–43.5% for BaP). However, the repeatability was good (RSDr of 8.78–9.96% for Ant and 6.43–11.9% for BaP), and the HorRat values were acceptable (1.56–1.94 for Ant and 1.10–1.45 for BaP). In all other cases, repeatability, reproducibility, and HorRat values were as follows: shrimp: RSDr 1.40–26.9%, RSDR 5.41–29.4%, HorRat: 0.22–1.34; mussel: RSDr 2.52–17.1%, RSDR 4.19–32.5%, HorRat: 0.17–1.13; and oyster: RSDr 3.12–22.7%, RSDR 8.41–31.8%, HorRat: 0.34–1.39. The results demonstrate that the method is fit-for-purpose to determine PAHs and their alkyl homologs in seafood samples. The method was approved by the Expert Review Panel on PAHs as the AOAC Official First Action Method 2014.08.