M astovska et al .: J ournal of AOAC I nternational V ol . 98, N o . 2, 2015  479

The blank shrimp samples were shipped frozen on dry ice with a material receipt document to be returned to the Study Directors. The test samples had to be stored in a freezer set to maintain at least –20 ± 10°C. The spiking solutions were to be stored in a refrigerator set to maintain 5 ± 3°C. ( Note : This modification of the shrimp test sample preparation protocol (as compared to mussel and oyster) was made (after consultations with the SPSC PAH Working Group and the AOAC Methods Committee on PAHs ) due to potential stability issues discovered during the practice sample analysis and follow-up experimentswith fortified shrimp samples stored at different conditions. 3-Methylchrysene (3-MC) had to be replaced by 6-methylchrysene (6-MC) in the spiking and calibration solutions for shrimp samples due to the unavailability of a 3-MC reference standard at the time of preparation and shipment of the new set of shrimp samples to the study participants.) During the laboratory qualification phase, the collaborators conducted the following seven steps. These steps were necessary because the Study Directors allowed the use of various GC/MS instruments, GC columns, silica SPE cartridges, and evaporation techniques and equipment. Therefore, performance-based criteria were developed to help laboratories optimize their GC/MS, SPE cleanup, and solvent evaporation conditions; check and eliminate potential PAH contamination in their reagent blanks; and become familiar with the method. Laboratory qualification and practice sample results had to be approved by the Study Directors before proceeding with the test sample analysis. Sixteen laboratories entered the qualification phase, but only 10 of them (listed in the Acknowledgments section) completed the qualification successfully and/or continued in the study. ( 1 ) The first step was a GC separation test where participants analyzed a composite PAH solution by GC/MS/MS to obtain a baseline separation of BaP and benzo[ e ]pyrene (concentration ratio of 1:5); at least 50% valley separation of anthracene and phenanthrene (concentration ratio 1:2.5, evaluated for the anthracene peak); and at least 50% valley separation for benzo[ b ]fluoranthene, benzo[ j ]fluoranthene, and benzo[ k ] fluoranthene (concentration ratio of 1:1:1). ( 2 ) The second step was a calibration range test where participants prepared calibration standards and obtained normalized calibration curves for the studied PAHs versus respective labeled internal standards ( 13 C-PAHs). Collaborators had to determine the linear range, test for carryover by injecting a solvent blank after the highest standard, and adjust injection conditions (such as injection volume, number of washes, syringe size, etc.) to achieve low detection limits, acceptable linearity for the tested concentration range, and minimum carryover. Coefficient of determination (r 2 ) values should be 0.990 or greater, and back-calculated concentrations of the calibration standards should not exceed ±20% of theoretical. For lower concentration levels, a limited calibration curve (without the higher-end concentration points) may be used for better accuracy. If a well characterized quadratic relationship occurs, then a best-fitted quadratic curve may be used for calibration. Otherwise, if the back-calculated concentrations exceed ±20% of theoretical, normalized signals of the nearest two calibration Laboratory Qualification

Collaborative Study


The purpose of this study was to evaluate the method’s intralaboratory and interlaboratory performance and submit the results to AOAC INTERNATIONAL for adoption as an Official Method for the determination of PAHs in seafood.

Study Design

This study evaluated the method performance for determination of 19 selected PAHs, including alkyl homologs relevant to an oil spill contamination ( see Table 1), in three seafood matrixes: shrimp, oysters, and mussels, with five different levels of BaP ranging from 2 to 50 µg/kg. Each matrix had a varying mixture of three different BaP levels (“low,” “mid,” and “high”). The other studied PAHs were added at varying levels from 2 to 250 µg/kg to mimic typical PAH patterns (Table 2). The fortified analytes in the 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. The AOAC official method guidelines for collaborative study procedures (3) were followed for the preparation of the study and data analysis. Blank mussel and oyster samples were homogenized with liquid nitrogen and tested in duplicate by an independent laboratory for potential contamination with the target PAHs. During homogenization, portions of the blank matrixes were spiked with 1,7-dimethylphenanthrene (1,7-DMP) at 40 and 80 µg/kg in the case of mussel and oyster, respectively. These were utilized as a homogenization check throughout the course of the study. The collaborators determined 1,7-DMP along with the other 18 analytes, which were spiked into 10 g sample portions placed in polypropylene centrifuge tubes by the study direction team. Five different spiking levels were made at varying PAH concentrations (Table 2), resulting in three different duplicate spiked samples/matrix in addition to a blank. Participants were supplied with the test samples ready for analysis labeled with unique identification numbers. All test samples were shipped frozen on dry ice with a material receipt document to be returned to the Study Directors. The test samples had to be stored in a freezer set to maintain at least –20 ± 10°C. Test samples were to be analyzed after completion of laboratory qualification and practice sample analysis. Blank shrimp matrix (peeled, without head and tail, and uncooked) was homogenized without the use of liquid nitrogen using a blender. After testing for potential contamination with the target PAHs, 10 g blank sample portions were placed in polypropylene centrifuge tubes, which were sent to study participants together with spiking solutions labeled with unique identification numbers. Using instructions provided by the Study Directors, participants fortified the blank shrimp samples themselves on the day of the analysis. Three different spiking levels were used at varying PAH concentrations (Table 2), resulting in three different duplicate spiked samples in addition to a blank (seven samples altogether). Test Sample Preparation

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