2. AOACRIChemContMethods-2018Awards

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

standards that enclose the analyte signal in the sample could be used to interpolate the analyte concentration. ( 3 ) The third step was a test of the solvent evaporation where participants determined absolute recoveries of both PAHs and 13 C-PAHs in two evaporation experiments (with three replicates each): ( a ) gentle evaporation of 5 mL of a PAH/ 13 C-PAH solution in ethyl acetate and reconstitution in isooctane and ( b ) gentle evaporation of 10 mL of a PAH/ 13 C-PAH solution in hexane–dichloromethane (3 + 1, v/v) and reconstitution in isooctane. The absolute recoveries of all analytes, including naphthalene, and 13 C-naphthalene had to be above 70%. ( 4 ) The fourth step was the determination of the elution profiles of PAHs and fat on silica gel SPE columns chosen for the PAH analysis by the laboratory. The silica gel columns could be prepared in-house using the procedure described in the method or could be obtained commercially from different vendors. The elution volume of 10 mL hexane–dichloromethane (3 + 1, v/v) specified in the ICT method (1) was optimized for the analysis of PAHs, PCBs, and PBDEs using the in-house prepared silica gel minicolumns for which the silica gel deactivation (5% water added) and storage are controlled by the laboratory. For commercially available silica gel SPE cartridges, however, the deactivation and storage can vary, potentially resulting in different amounts of water in the silica thus potentially different retention characteristics. Therefore, it is important to test the elution profiles of PAHs and fat and determine the optimum volume of the elution solvent to ensure adequate analyte recoveries and fat cleanup. The PAH elution profile was determined by applying 1 mL of a PAH in hexane solution to the silica cartridge, collecting fractions of hexane–dichloromethane (3 + 1, v/v) eluting from the cartridge, exchanging the fractions to 0.5 mL isooctane, and analyzing them by GC/MS. The fat elution profile was checked gravimetrically by applying 1 mL of hexane containing 100 mg of fat (pure fish oil) onto the silica cartridge, collecting the optimum elution fraction determined for PAHs and three consecutive 1 mL fractions, and evaporating them to dryness. ( 5 ) The fifth step was a reagent (procedure) blank test where participants determined concentrations of the target PAHs in three replicates of reagent (procedure) blank that was prepared the same way as the samples, except that 10 mL of water was used instead of the sample. The concentrations of all analytes in the reagent blanks had to be below the concentrations in the lowest calibration level standard. For naphthalene, levels below the second lowest calibration standard (equivalent to 5 ng/g of naphthalene in the sample) were still acceptable if the source of contamination could not be eliminated, such as by selection of a silica gel SPE column from a different vendor (or preparation of silica gel columns in-house), heating of glassware, addition of a hydrocarbon trap to the nitrogen lines used for solvent evaporation, etc. ( 6 ) The sixth step was a low-level spike test where collaborators prepared and analyzed seven spiked samples using blank shrimp matrix and a mixed PAH spiking solution that were both supplied to them. The samples were spiked at PAH concentrations equivalent to the second lowest calibration level (1 µg/kg for BaP, which is a fitness-for-purpose LOQ requirement established for the study) to test instrument sensitivity and method precision. The shrimp matrix had to be stored in a freezer set to maintain at least –20 ± 10°C. The

mixed PAH spiking solution was to be stored in a refrigerator set to maintain 5 ± 3°C. ( 7 ) The seventh step was the analysis of practice samples. Three practice samples were supplied to the participants. Two of the three samples were shrimp blank matrix already spiked with two different mixed PAH solutions (BaP levels of 2–50 µg/kg, other PAHs at 2–250 µg/kg). The third sample was the National Institute of Standards and Technology Standard Reference Material 1974b, which is a mussel matrix with certified concentrations of incurred PAHs and other organic contaminants. All practice samples were shipped frozen on dry ice and had to be stored in a freezer set to maintain at least –20 ± 10°C. The method uses a mixture of isotopically labeled 13 C-PAH surrogate standards that were added at 5 µg/kg to the samples prior to the extraction process. Quantification was based on calibration of analyte signals (peak areas or heights) divided by signals of respective 13 C-labeled internal standards plotted versus analyte concentrations. Eight concentration levels were used for the calibration, corresponding to 0.5, 1, 2, 5, 10, 20, 50, and 100 µg/kg for BaP and other lower level PAHs, and to 1.25, 2.5, 5, 12.5, 25, 50, 125, and 250 µg/kg for higher level PAHs, except for naphthalene that was present at levels corresponding to 2.5, 5, 10, 25, 50, 100, 250, and 500 µg/kg. Values of r 2 had to 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 three higher-end concentration points) was used for better accuracy. In addition to reporting r 2 values, back-calculated calibration standard concentrations, and analyte concentrations, the collaborators were also required to report ion ratios as a means of verifying identification of the analyte peaks. A solvent (isooctane) blank was injected before and after each calibration set. Reagent (procedural) blanks were analyzed with each set of samples. During homogenization, portions of the blank mussel and oyster matrixes were spiked with 1,7-DMP, which served as a homogenization check of the sample processing step. Participants supplied PAH and 13 C-PAH signals (peak areas or heights) in test samples, calibration standards, and blanks and other parameters as described above in Quality Assurance in Excel forms created by the Study Directors. They also had to provide details about their GC and MS instruments and method conditions, evaporation equipment and conditions, and silica gel SPE cartridge and optimum elution volume. Participants were asked to record all observations and any potential method deviations, investigate any potential unreasonable results (caused by, e.g., incorrect calculations and arithmetic errors, use of wrong units, transposition errors, incorrect standard preparation or contamination), and have all the results and Quality Assurance Data Reporting