M
astovska
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
98, N
o
. 2, 2015
487
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.
(
10
) Condition a silica SPE column (1 g silica gel with
approximately 0.2 g of muffled anhydrous sodium sulfate on
the top) with 6 mL hexane–dichloromethane (3 + 1, v/v) and
4 mL hexane.
(
11
) Apply the extract in hexane onto the silica SPE cartridge.
(
12
) Elute with hexane–dichloromethane (3 + 1, v/v) using
volume determined for the given silica gel SPE cartridges
from the elution profiles of target analytes and fat, which are
dependent on the silica deactivation,
see Note
(
4
) below. Collect
the eluent.
(
13
) Add 0.5 mL isooctane (and 1-2 mL ethyl acetate) to
the eluent as a keeper and gently evaporate down to 0.5 mL to
remove hexane and dichloromethane from the final extract.
(
14
) Transfer the final extract into an autosampler vial for the
GC/MS analysis.
Notes
: (
1
) The fat capacity of the 1 g silica gel SPE column
is approximately 0.1 g. If the 5 mL ethyl acetate extract aliquot
contains more than 0.1 g fat, it is necessary to use a smaller
aliquot volume to avoid sample breakthrough during the
cleanup step.
(
2
) Ethyl acetate should not be present in the extract applied
to the silica cartridge because it can affect the extract polarity,
thus potentially retention of fat and analytes on the silica gel.
The coextracted fat and 50 µL isooctane act as keepers during
the first evaporation step (step
8
), thus the evaporation should
be conducted gently until there is no significant change in the
volume, i.e., until only the isooctane and coextracted fat are left
in the evaporation tube or flask.
(
3
) Addition of 1-2 mL ethyl acetate to the eluent in step
13
is recommended for a better control of the evaporation process
and higher absolute recoveries of volatile PAHs.
(
4
) The deactivation and storage of silica gel SPE cartridges
can vary, potentially resulting in different amounts of water in
the silica, thus its 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
following procedure is recommended:
(
a
) Prepare a PAH solution in hexane by combining
50 µL of the Working PAH Solution A and 1 mL hexane
in a vial. Mix well and apply onto a silica SPE column (1 g
silica gel with approximately 0.2 g of muffled anhydrous
sodium sulfate on the top), which was conditioned with 6 mL
hexane–dichloromethane (3 + 1, v/v) and 4 mL hexane.
(
b
) Elute with 10 mL hexane–dichloromethane (3 + 1, v/v),
Table 2014.08D. (
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
9
18
20.0
80.1
1.0
2.2
4.8
10.7
0.37
Naph
9
18
71.0
88.7
5.3
9.4
7.5
13.2
0.55
9
18
106.2
84.9
7.3
14.7
6.9
13.9
0.62
8
16
193.9
86.2
6.0
29.8
3.1
15.4
0.75
Phe
9
18
41.6
83.2
3.0
5.5
7.2
13.2
0.51
9
18
80.3
80.3
6.1
10.7
7.6
13.3
0.57
8
16
203.9
81.6
9.5
22.5
4.7
11.0
0.54
Pyr
9
18
34.0
85.1
2.2
3.3
6.4
9.8
0.37
8
16
63.2
84.3
2.2
5.3
3.5
8.4
0.35
9
18
163.4
81.7
8.0
16.6
4.9
10.2
0.48
Figure 2014.08A. Flow chart of the method for determination of PAHs in seafood using GC/MS.
10 g of homogenized sample
- Add
13
C-PAH mixture, vortex, equilibrate (15 min)
Extraction:
- Add 5 mL (or 10 mL) water and 10 mL EtOAc, shake (1 min)
- Add 4 g anh. MgSO4 and 2 g NaCl, shake (1 min), centrifuge
- Evaporate 5 mL aliquot of extract, reconstitute in 1 mL hexane
Silica-SPE clean-up:
-
Condition 1g silica with 6 mL hexane:DCM (3:1,
v/v
) and 4 mL
hexane
-
Apply sample
-
Elute with 10 mL of hexane:DCM (3:1,
v/v
)
GC-MS(/MS) analysis
Figure 2014.08A. Flow chart of the method for determination of
PAHs in seafood using GC/MS.