504  M astovska et al . : J ournal of AOAC I nternational V ol . 98, N o . 2, 2015

and confirmed the lower recoveries for Ant and BaP. This collaborator also made another interesting observation related to the color difference between extracts obtained in the first set (all dark green) and the second set (the dark green extract was produced only for the blank sample), whereas all extracts of fortified samples were yellow-brown. This observation, which was later confirmed by additional study participants, indicates matrix changes caused by the presence of PAHs and accompanied by selective losses of Ant and BaP. In addition to these two analytes, BaA also showed lower recoveries (around 70%) in oysters when compared to the rest of the studied PAHs. Furthermore, a closer examination of the results obtained for Ant, BaP, and BaA in mussel also show somewhat lower mean recoveries of these three analytes (around 80%) when compared to the other analytes. These mean results do not include data obtained for BaP in mussel test samples by Laboratory No. 6, which analyzed the test samples about a year later than most other participants. All Laboratory No. 6 BaP results in mussel test samples showed very good repeatability within the duplicates but were eliminated as Grubbs’ test outliers because they were significantly lower than the results obtained by other laboratories. No other data obtained by Laboratory No. 6 were identified as outliers using the Cochran or Grubbs’ tests. Figure 1 provides structures of Ant, BaA, and BaP showing that these PAHs contain the same moiety in terms of the linear 3-ring (anthracene) structure. This structural commonality and similar degradation behavior indicate that they could be substrates for the same enzyme(s). The significant differences in recoveries obtained for samples stored at –20°C versus –70°C (Tables 12 and 13, respectively) represent more supporting evidence for an enzymatic degradation being the most probable cause for the lower recoveries observed for these analytes in oyster (and mussel) test samples. To prevent degradation of these analytes in seafood matrixes during long-term storage, the Study Directors recommend storing homogenized samples at –70°C or lower. Unfortunately, this was not a feasible requirement for this collaborative study because the majority of the collaborating laboratories did not have this storage capability. The Study Directors wish to thank the SPSC PAH Working Group (chaired by Gina Ylitalo from National Oceanic and Atmospheric Administration), the AOAC Methods Committee on PAHs (chaired by Tom Phillips, Maryland Department of Agriculture, Annapolis, MD), and AOAC INTERNATIONAL staff for the discussions about the study design, analyte selection, and stability issues as well as for the overall support provided to the collaborative study. Special acknowledgment goes to Jack Cochran (Restek Corp., Bellefonte, PA) for useful discussions about the PAH GC/MS analysis. John Schmitz and Jack Jabusch (Covance Laboratories, Madison, WI) and Lucie Drabova and Jana Pulkrabova (ICT, Prague, Czech Republic) are acknowledged for technical advice and support during the preparation and the entire course of the study. The Study Directors thank the Nutritional Chemistry and Food Safety business unit at Covance Laboratories for supplying and preparing PAH standards and spiking solutions; obtaining Acknowledgments

to the blind duplicates) in all seven test samples was 70.5 µg/kg (RSD = 16.3%, n = 56), which corresponds to mean recovery of 88.1%. These results indicate that the mussel and oyster samples sent to the study participants had good homogeneity. 1,7-DMP was also added to all shrimp test samples at 20 µg/kg during the fortification step conducted by the study participants. The mean concentration value was 22.1 µg/kg (RSD = 19.0%, n = 63), corresponding to mean recovery of 111%. Statistical results for 1,7-DMP obtained in blind duplicate samples are summarized together with the other analytes in Tables 2014.08B–D . Tables 2014.08B–D provide statistical results obtained for the studied analytes at three different concentration levels in shrimp, mussel, and oyster after elimination of statistical outliers (highlighted in Tables 3–11). Eight to 10 valid results were obtained for the majority of determinations. Mean recoveries of all tested analytes at the total of 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 BaA fortified at 25 µg/kg in oyster (RSD r : 5.84%, RSD R : 21.1%) and lower mean recoveries for 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 ( see the discussion about degradation issues below), which also resulted in lower reproducibility (RSD R values in the range of 44.5–64.7% for Ant and 40.6–43.5% for BaP). However, the repeatability was good (RSD r 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: ( 1 ) Shrimp: RSD r 1.40–26.9%, RSD R 5.41–29.4%, HorRat 0.22–1.34; ( 2 ) Mussel: RSD r 2.52–17.1%, RSD R 4.19–32.5%, HorRat 0.17–1.13; and ( 3 ) Oyster: RSD r 3.12–22.7%, RSD R 8.41–31.8%, HorRat 0.34–1.39. Overall, the results of the collaborative study demonstrate that the method is fit-for-purpose to determine PAHs and their alkyl homologs in seafood samples. The Study Directors reported problems with lower recoveries for Ant and BaP in oyster samples stored at –20°C to the SPSC PAH Working Group and the AOAC Methods Committee on PAHs when they discovered a significant difference between results for these two analytes obtained in two different participating laboratories storing the samples at two different freezer temperatures of –20 and –70°C ( see recovery results in Tables 12 and 13, respectively). Due to the limited availability and cost of oyster samples, it was decided to continue with the study and not proceed with preparation of new study test samples that would be fortified by collaborators on the day of the analysis as was done for shrimp ( Note : in the case of shrimp, overall lower recoveries were obtained for all studied PAHs depending on the shrimp sample storage conditions). The laboratory storing samples at –20°C analyzed another set of oyster samples about 1.5 months after the first set Degradation Issues

Made with FlippingBook flipbook maker