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(
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),
collecting 0.5 mL elution fractions in 20 evaporation tubes or flasks.
Add 0.5 mL isooctane to each elution fraction and evaporate down
to 0.5 mL using the optimized evaporation conditions. Analyze
each fraction by GC/MS.
(
c
) Determine PAH elution profile by plotting analyte response
(peak area or height) in a given fraction normalized to the sum
of analyte responses in all tested fractions vs the elution volume.
See
Figure
2014.08B
for an example of a PAH elution profile. It is
recommended to add an additional 0.5 mL on top of the determined
elution fraction (corresponding to 100% recovery) as a safety
margin ensuring good analyte recoveries in routine practice. This
would result in the optimum elution volume of 7 mL for the silica
cartridge tested in Figure
2014.08B
.
(
d
) To check the effectiveness of fat removal, dissolve 100 mg
pure fish oil (or any suitable fat) in 1 mL hexane and apply it
onto the silica gel cartridge, which was conditioned with 6 mL
hexane–dichloromethane (3 + 1, v/v) and 4 mL hexane. Elute with
the optimum elution volume of hexane–dichloromethane (3 +
1, v/v), which was determined in the previous step (e.g., 7 mL
for the example in Figure
2014.08B
). Collect this fraction in an
evaporation tube or flask, which empty weight (after heating in
an oven to remove moisture) was recorded to four decimal places
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
Figu 2014.08A. Flow chart of the method for
determination of PAHs in seafood using GC/MS.
Candidates for 2016 Method of the Year
226