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480 Mastovska et al.: Journal of AOAC International Vol. 98, No. 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
13C-PAHs in two evaporation experiments (with three replicates
each): (a) gentle evaporation of 5 mL of a PAH/13C-PAH
solution in ethyl acetate and reconstitution in isooctane and
(b) gentle evaporation of 10 mL of a PAH/ 13C-PAH solution
in hexane–dichloromethane (3 + 1, v/v) and reconstitution in
isooctane. The absolute recoveries of all analytes, including
naphthalene, and 13C-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.
Quality Assurance
The method uses a mixture of isotopically labeled 13C-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 13C-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 r2 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.
Data Reporting
Participants supplied PAH and 13C-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
Candidates for 2016 Method of the Year
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