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© 2015 AOAC INTERNATIONAL
(
c
)
WS3 (0.01 mg/L)
.—Dilute 500 µL of WS2 (0.1 mg/L) to
5 mLwith deionized water in a calibrated volumetric flask. Transfer
into a 15 mL polypropylene tube. Make fresh daily.
(
2
)
Internal standard
.—
Internal standard working solution
(ISWS; 0.5 mg/L)
.—Dilute 1000 µL of
13
C
2
D
2
fluoroacetic acid
intermediate standard (50 mg/L) to 100 mL with deionized water
in a calibrated volumetric flask. Pipet aliquots of the ISWS into
15 mL polypropylene tubes. Store in the freezer at –10°C or
below. A thawed and opened tube of ISWS can be stored for up to
2 months in a refrigerator between 2–8°C provided it is resealed
and immediately refrigerated after each use.
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 µLWS2 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.
Table 2015.02D. Instrument parameters for AB Sciex
LC-MS/MS system
Parameter
Value
HPLC column
Agilent XDB-C18,
100
×
4.6 mm
×
1.8 µm
Column temperature
60°C
Autosampler temperature
10°C
Flow rate
1 mL/min
Injection volume
5 µL
Run time
4 min
Ionization mode
Electrospray
Polarity
Negative
Curtain gas (CUR)
30 psi
Source temp. (TEM)
750°C
Ion source gas 1 (GS1)
60 psi
Ion source gas 2 (GS2)
60 psi
Ion spray voltage (IS)
–4500 V
Collision gas (CAD)
Medium (8)
Entrance potential (EP)
–10 V
Table 2015.02C. HPLC solvent gradient
Time
% A
(10 mM NH
4
Ac in H
2
O)
% B
(10 mM NH
4
Ac in 97% ACN)
0
80
20
2.50
0
100
3.00
0
100
3.01
80
20
4.00
80
20
Candidates for 2016 Method of the Year
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