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M
astovska
et al
.
:
J
ournal of
AOAC I
nternational
V
ol
. 98, N
o
. 2, 2015
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.
H. Calculations
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);
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.
Results and Discussion
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
Table 2014.08I. PAH analytes and corresponding
13
C-PAHs used for PAH signal normalization
Analyte
13
C-PAH used for signal normalization
Anthracene
Anthracene (
13
C
6
)
Benz[
a
]anthracene
Benz[
a
]anthracene (
13
C
6
)
Benzo[
a
]pyrene
Benzo[
a
]pyrene (
13
C
4
)
Benzo[
b
]fluoranthene
Benzo[
b
]fluoranthene (
13
C
6
)
Benzo[
g,h,i
]perylene
Benzo[
g,h,i
]perylene (
13
C
12
)
Benzo[
k
]fluoranthene
Benzo[
k
]fluoranthene (
13
C
6
)
Chrysene
Chrysene (
13
C
6
)
Dibenz[
a,h
]anthracene
Dibenz[
a,h
]anthracene (
13
C
6
)
Fluoranthene
Fluoranthene (
13
C
6
)
Fluorene
Fluorene (
13
C
6
)
Indeno[1,2,3-
cd
]pyrene
Indeno[1,2,3-
cd
]pyrene (
13
C
6
)
Naphthalene
Naphthalene (
13
C
6
)
Phenanthrene
Phenanthrene (
13
C
6
)
Pyrene
Pyrene (
13
C
6
)
1-Methylnaphthalene
Naphthalene (
13
C
6
)
2,6-Dimethylnaphthalene
Phenanthrene (
13
C
6
)
1-Methylphenanthrene
Phenanthrene (
13
C
6
)
1,7-Dimethylphenanthrene
Phenanthrene (
13
C
6
)
3-Methylchrysene
Chrysene (
13
C
6
)