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© 2015 AOAC INTERNATIONAL
Containing: Acenaphthene (
13
C
6
, 99%), acenaphthylene (
13
C
6
,
99%), anthracene (
13
C
6
, 99%), benz[
a
]anthracene (
13
C
6
, 99%),
benzo[
b
]fluoranthene (
13
C
6
, 99%), benzo[
k
]fluoranthene (
13
C
6
,
99%), benzo[
g,h,i
]perylene (
13
C
12
, 99%), benzo[
a
]pyrene (
13
C
4
,
99%), chrysene (
13
C
6
, 99%), dibenz[
a,h
]anthracene (
13
C
6
, 99%),
fluoranthene (
13
C
6
, 99%), fluorene (
13
C
6
, 99%), indeno[1,2,3-
cd
]
pyrene (
13
C
6
, 99%), naphthalene (
13
C
6
, 99%), phenanthrene (
13
C
6
,
99%), and pyrene (
13
C
6
, 99%).
E. Preparation of Standard Solutions
(
a
)
Individual stock solutions.—
Prepare individual PAH stock
solutions at approximately 1000 or 2500 µg/mL in toluene.
(
b
)
Mixed stock standard solution.—
Use analyte individual
stock solutions to obtain a mixed solution of each PAH at
10 µg/mL (for benzo[
a
]pyrene and other low-level PAHs) or
25 µg/mL (for chrysene and other higher-level PAHs) or 50 µg/mL
(for naphthalene) in isooctane.
See
Table
2014.08E
for analyte
concentrations in the mixed stock standard solution.
(
c
)
Working PAH Solution A.—
Accurately transfer 0.5 mL of the
mixed stock standard solution into a 5 mL volumetric flask and
dilute to volume with isooctane.
(
d
)
Working PAH Solution B.—
Accurately transfer 0.5 mL of the
Working PAH Solution A into a 5 mL volumetric flask and dilute to
volume with isooctane.
(
e
)
Internal standard solution.—
Prepare 1 µg/mL solution of
13
C-PAHs in isooctane by 5-fold dilution of the 5 µg/mL EPA 16
13
C-PAHs cocktail with isooctane.
(
f
)
Calibration standard solutions.—
Prepare eight levels of
calibration standard solutions (1 mL each) in 2 mL amber screw-
cap vials. It is recommended to distribute small portions (enough
for a single injection) of the calibration standard solutions into
multiple crimp-top vials with 100 µL deactivated glass inserts.
See
Table
2014.08A
for analyte concentrations in the calibration
standards and Table
2014.08F
for the dilution scheme.
(
1
)
For level 1 calibration standard
.—Accurately transfer
50 µL of the Working PAH Solution B into the vial and add 50 µL
of the 1 µg/mL
13
C-PAHs solution and 900 µL isooctane. Cap the
vial and vortex mix briefly.
(
2
)
For level 2 calibration standard
.—Accurately transfer
100 µL of the Working PAH Solution B into the vial and add 50 µL
of the 1 µg/mL
13
C-PAHs solution and 850 µL isooctane. Cap the
vial and vortex mix briefly.
(
3
)
For level 3 calibration standard
.—Accurately transfer
200 µL of the Working PAH Solution B into the vial and add 50 µL
of the 1 µg/mL
13
C-PAHs solution and 750 µL isooctane. Cap the
vial and vortex mix briefly.
(
4
)
For level 4 calibration standard
.—Accurately transfer
500 µL of the Working PAH Solution B into the vial and add 50 µL
of the 1 µg/mL
13
C-PAHs solution and 450 µL isooctane. Cap the
vial and vortex mix briefly.
(
5
)
For level 5 calibration standard
.—Accurately transfer
100 µL of the Working PAH Solution A into the vial and add 50 µL
of the 1 µg/mL
13
C-PAHs solution and 850 µL isooctane. Cap the
vial and vortex mix briefly.
(
6
)
For level 6 calibration standard
.—Accurately transfer
200 µL of the Working PAH Solution A into the vial and add 50 µL
of the 1 µg/mL
13
C-PAHs solution and 750 µL isooctane. Cap the
vial and vortex mix briefly.
(
7
)
For level 7 calibration standard
.—Accurately transfer
500 µL of the Working PAH Solution A into the vial and add 50 µL
of the 1 µg/mL
13
C-PAHs solution and 450 µL isooctane. Cap the
vial and vortex mix briefly.
(
8
)
For level 8 calibration standard
.—Accurately transfer
100 µL of the mixed stock standard solution into the vial and add
50 µL of the 1 µg/mL
13
C-PAHs solution and 850 µL isooctane. Cap
the vial and vortex mix briefly.
F. Extraction and Cleanup Procedure
(
1
) Add 50 µL of the 1 µg/mL
13
C-PAHs solution to 10 ± 0.1 g of
thoroughly homogenized seafood sample in a 50 mL polypropylene
centrifuge tube.
(
2
) Vortex sample for 15 s and let equilibrate for 15 min.
(
3
) Add 5 mL (10 mL in the case of shrimp) of purified water
and 10 mL ethyl acetate.
(
4
) Shake tube vigorously by hand for 1 min.
(
5
) Add 4 g of muffled anhydrous magnesium sulfate and 2 g
sodium chloride, and seal the tube well (ensure that powder does
not get into the screw threads or rim of the tube).
(
6
) Shake tube vigorously by hand for 1 min, ensuring that
crystalline agglomerates are broken up sufficiently during shaking.
(
7
) Centrifuge tube at >1500 rcf for 10 min.
(
8
) Take a 5 mL aliquot of the upper ethyl acetate layer, add
50 µL isooctane as a keeper, and gently evaporate all ethyl acetate
until only isooctane and co-extracted sample fat are left.
(
9
) Reconstitute in 1 mL hexane.
Table 2014.08C. (
continued
)
PAH
No. of
laboratories
No. of
replicates
Mean
concn, µg/kg
Mean
recovery, % s
r
, µg/kg
s
R
, µg/kg RSD
r
, % RSD
R
, % HorRat
10
20
17.9
89.3
0.9
1.6
4.9
8.8
0.30
Naph
9
18
23.7
94.6
1.9
4.0
8.1
17.1
0.61
10
20
105.9
84.7
10.1
26.7
9.5
25.2
1.12
8
16
146.7
91.7
7.2
19.2
4.9
13.1
0.61
Phe
8
16
14.5
96.8
0.8
0.9
5.3
6.1
0.20
8
16
93.1
93.1
3.9
8.5
4.1
9.2
0.40
8
16
160.8
91.9
5.8
13.0
3.6
8.1
0.38
Pyr
10
20
14.2
94.6
0.7
1.3
5.0
9.3
0.31
10
20
71.5
95.4
2.9
7.0
4.0
9.8
0.41
10
20
116.5
93.2
4.5
9.6
3.8
8.3
0.37
Candidates for 2016 Method of the Year
224