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Joseph et al.:
J
ournal of
AOAC I
nternational
V
ol.
98, N
o
. 3, 2015
1125
E. Sampling and Sample Preparation
Preparation of test portion
.—Accurately weigh 2.5 ± 0.03 g
of room temperature sample into a labeled 50 mL polypropylene
tube. In addition to the analytical samples, there are four
recovery samples per batch, a reagent blank, and an extra blank
for the matrix standard.
F. Procedure
(
a
)
Fortification
.—(
1
)
Analyte
.—Fortify the recoveries as
shown in Table
2015.02A
using the working solutions prepared
in
D
(
d
).
Note
: Do not add any WS to the matrix blank to be used
for the matrix standard.
(
2
)
Internal standard
.—Add 40 µL ISWS to all unknown
and recovery samples.
Note
: Do not add any ISWS to the matrix
blank to be used for the matrix standard.
(
3
) Allow test portions to equilibrate for 10 min at room
temperature.
(
b
)
Extraction
.—(
1
) Place the resin chromatography
columns onto a vacuum manifold and fill with 1.4 (±0.2) mL
resin. Add 2.5 mL deionized water above the resin bed and close
stopcock. Fit suitable reservoirs above the columns.
(
2
) To each test portion add 5 mL water and briefly shake
vigorously by hand, cap, and then shake tubes at medium speed
on a reciprocating shaker for 5 min to dissolve. Variation to this
procedure may be required for atypical matrixes.
(
3
) Add 10 mL acetone to each tube and briefly shake
vigorously by hand followed by 2 min on a reciprocating shaker
at medium speed.
(
4
) Centrifuge at 4200 ×
g
RCF for 10 min.
(
5
) Carefully pour the top solvent layer into the reservoirs
above the resin, taking care not to transfer any precipitate.
(
6
) Allow samples to pass through the resin columns under
gravity or gentle vacuum, if required.
(
7
) After samples have passed through the resin columns,
remove the reservoirs and wash the resin columns with 1 mL
of 0.2 M hydrochloric acid. Close stopcock. Do not allow the
resin to dry.
(
8
) Place 15 mL polypropylene tubes beneath each resin
column. Elute samples with one 5 mL volume of 0.2 M
hydrochloric acid at about 30 drops/min. Remove residual
hydrochloric acid solution into the collecting tubes under
vacuum.
(
9
) To the matrix standard tube only,
add 125 µL WS2 and
40 µL ISWS, cap, and vortex mix.
(
10
) To all tubes add 1.25 mL of 20 mg/mL 3-nitroaniline
and 0.25 mL of 100 mg/mL EDAC solution followed by 0.5
mL of 2 M potassium hydroxide and 1 mL of 0.05 M potassium
dihydrogen phosphate buffer. Cap and mix.
(
11
) Place tubes in a 40 ± 2°C water bath for 20 min.
(
12
) Remove tubes and cool to room temperature.
(
13
) Set up a vacuum manifold with Oasis HLB, 60 mg,
3 mL cartridges.
(
14
) Condition the cartridge with 1 mL methanol. Close the
stopcock when the methanol reaches the top frit.
(
15
) Load a portion of the derivatized extract onto the
conditioned SPE cartridge.
(
16
) Place an adapter and 10 mL reservoir on top of the
cartridge.
(
17
) Transfer the remaining derivatized extract into the
reservoir and open the stopcock. Allow to drip slowly to waste
at about 30–40 drops/min.
(
18
) When the extract has passed through the cartridge,
remove the adapter and reservoir.
(
19
) Wash the cartridge with 2 mL 25% (v/v) sulfuric acid,
1 mL deionized water, 1 mL 0.1 M sodium hydrogen carbonate,
and a further 2 mL deionized water to waste.
(
20
) Dry cartridge by applying full vacuum for 5 min.
(
21
) Place 15 mL polypropylene tubes beneath each SPE.
Elute the derivatized extract with 2 × 2.5 mL TBME–
n
-hexane
(70 + 30, v/v) into the tubes.
(
22
) Dry the cartridge by briefly applying a full vacuum.
(
23
) Check tubes for remaining water. There should be
minimal water present. Presence of more than about 50 µL
water would indicate inadequate vacuum.
(
24
) Add approximately 200 mg sodium sulfate, anhydrous,
to each tube and vortex mix.
(
25
) Centrifuge at 2400 ×
g
RCF for 1 min.
Table 2015.02F. Relative retention time (RRT) and limits of acceptance
Compound (3-nitroaniline derivative of analyte)
Monitored compounds
RRT
Acceptance limit
a
2-Fluoro-3
ʹ
-nitroacetanilide
Analyte/internal standard
1.004
b
RRT ± 2.5%
a
See
reference 2.
b
Representative relative retention time. These values are indicative and should be measured for each individual batch.
Table 2015.02E. Performance values of analytes
a
Compound
LOD, µg/kg LOQ, µg/kg LOR, µg/kg Within-day CV
Between-day CV
(WLR)
U
(for 95% CI)
Recovery, %
(SD)
Fluoroacetic acid
0.028
0.085
1.0
b
8.8
9.1
18
97
c
(8.8)
13
C
2
D
2
Fluoroacetic acid
NA
NA
NA
NA
NA
NA
70
d
(12)
a
LOD = Limit of detection; LOQ = limit of quantification; CV = coefficient of variation; WLR = within-laboratory reproducibility; U = uncertainty of
measurement with a 95% confidence interval; SD = standard deviation.
b
Limit of reporting (LOR) set according to New Zealand maximum permitted residue limits.
See
reference 1.
c
Relative recovery.
d
Absolute recovery.
244