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

X 13C-PAH is the amount of the corresponding 13 C-PAH added to the sample (in ng); and m is the sample weight (in g). Based on the method procedure and preparation of the calibration standard solutions, c 13C-PAH is 50 µg/L, X 13C-PAH is 50 ng, and m for the test samples is 10 g. In the collaborative study, eight concentration levels were used for the calibration, corresponding to 5, 10, 20, 50, 100, 200, 500, and 1000 µg/L for benzo[ a ]pyrene and other lower-level PAHs, to 12.5, 25, 50, 125, 250, 500, 1250, and 2500 µg/L for higher-level PAHs, except for naphthalene that was present at 25, 50, 100, 250, 500, 1000, 2500, and 5000 µg/L. Coefficients of determination (r 2 ) 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 standards that enclose the analyte signal in the sample can be used to interpolate the analyte concentration in the final extract. Laboratory Qualification Phase The analysis of PAHs poses several difficulties due to their physicochemical properties and occurrence in the environment and various materials that can lead to contamination issues. PAH properties, such as their volatility, polarity, and structure, affect their GC separation, MS determination/identification, and recoveries during solvent evaporation and silica SPE steps. To allow for flexibility and the use of various instruments, equipment, and columns, the Study Directors did not want to prescribe the use of a specific GC/MS instrument, GC column and separation conditions, silica SPE cartridge, and evaporation technique, equipment, or conditions. For this reason, they developed performance-based criteria for the GC/MS analysis (including separation of critical PAH pairs/groups, calibration range, or carryover), optimum elution volume in the SPE step (based on the elution profiles of PAHs and fat dependent on the silica deactivation), and evaporation conditions (to avoid significant loses of volatile PAHs, mainly naphthalene). These criteria were part of the laboratory qualification phase to help laboratories optimize conditions independent of their instrument/equipment choice or availability. This was also a very important consideration for the future implementation of the method in other laboratories. Another essential step in the laboratory qualification phase involved check of reagent blanks for potential PAH contamination. 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. Some laboratories found that their reagent Results and Discussion

Table 2014.08I. PAH analytes and corresponding 13 C-PAHs used for PAH signal normalization Analyte

13 C-PAH used for signal normalization

Anthracene ( 13 C 6 )


Benz[ a ]anthracene ( 13 C 6 ) Benzo[ a ]pyrene ( 13 C 4 ) Benzo[ b ]fluoranthene ( 13 C 6 ) Benzo[ g,h,i ]perylene ( 13 C 12 ) Benzo[ k ]fluoranthene ( 13 C 6 )

Benz[ a ]anthracene

Benzo[ a ]pyrene

Benzo[ b ]fluoranthene Benzo[ g,h,i ]perylene Benzo[ k ]fluoranthene

Chrysene ( 13 C 6 )


Dibenz[ a,h ]anthracene ( 13 C 6 )

Dibenz[ a,h ]anthracene

Fluoranthene ( 13 C 6 )


Fluorene ( 13 C 6 )


Indeno[1,2,3- cd ]pyrene ( 13 C 6 )

Indeno[1,2,3- cd ]pyrene

Naphthalene ( 13 C 6 ) Phenanthrene ( 13 C 6 )

Naphthalene Phenanthrene

Pyrene ( 13 C 6 )


Naphthalene ( 13 C 6 ) Phenanthrene ( 13 C 6 ) Phenanthrene ( 13 C 6 ) Phenanthrene ( 13 C 6 )

1-Methylnaphthalene 2,6-Dimethylnaphthalene 1-Methylphenanthrene 1,7-Dimethylphenanthrene

Chrysene ( 13 C 6 )


of the instrument. Verify identification of the analyte peaks by comparing the ion ratios of contemporaneously analyzed calibration standards, which have been analyzed under the same conditions. ( c ) Injection sequence. —Bracket the seven test samples with two sets of calibration standards. Inject solvent blanks after the calibration level 8 (highest) standard and after the samples. In addition, analyze a reagent blank with each set of samples. Inject only once from each vial, thus preventing potential losses of volatile PAHs and/or contamination. Quantification is based on linear least-squares calibration of analyte signals ( S PAH ) divided by signals ( S 13C-PAH ) of corresponding 13 C-labeled internal standards ( see Table  2014.08I ) plotted versus analyte concentrations. Peak areas are generally preferred as signals used for the quantification, but peak heights should be used for peaks that are not well resolved, such as in the case of anthracene and phenanthrene. The analyte concentrations in the final extract ( c PAH , µg/L) are determined from the equation: c PAH = [( S PAH / S 13C-PAH ) – b ]/ a where a is the slope of the calibration curve and b is the y -intercept. The concentration of PAHs in the sample ( C , µg/kg) is then calculated: C = ( c PAH / c 13C-PAH ) × ( X 13C-PAH / m ) where c 13C-PAH is the concentration of the corresponding 13 C-PAH in the calibration standard solutions (in µg/L); H. Calculations

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