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© 2015 AOAC INTERNATIONAL
Add 50 µL of the IS working solution 0.2 µg/mL,
C
(
g
). Mix
thoroughly and make sure that the spiked volume is totally absorbed
by the matrix. This spike corresponds to 10 µg/kg equivalent-in-
sample concentration of IS.
(
2
)
Liquid sample
.—Into a 15 mL polypropylene Falcon tube,
weigh 5.0 ± 0.1 g of liquid sample,
D
(
c
).
Add 250 µL of the IS working solution 0.2 µg/mL,
C
(
g
). Mix
thoroughly and make sure that the spiked volume is totally absorbed
by the matrix. This spike corresponds to 10 µg/kg equivalent-in-
sample concentration of IS.
(
b
)
Extraction procedure
.—To the test portion prepared as
described in
E
(
a
)(
1
) or
E
(
a
)(
2
), add 8 mL acetonitrile. Mix
thoroughly. Place onto a GenoGrinder shaker and shake for 1.5 min
at 1500 rpm.
Centrifuge at 4000 ×
g
at room temperature for 5 min and
transfer the supernatant (approximately 9 to 10 mL) into a 50 mL
Falcon tube.
Add 10 mL hexane. Place onto a GenoGrinder shaker and shake
for 1.5 min at 1500 rpm.
Centrifuge at 4000 ×
g
at room temperature for 5 min. Pipet the
upper hexane phase and discard it to waste.
Add 100 µL of concentrated sulfuric acid (H
2
SO
4
) to the solution
containing the analyte. Mix thoroughly. The resulting pH must be
≤1 to have the analyte in its acidic form (pKa of fluoroacetic acid
is 2.39).
Add a buffer salt mixture (Agilent QuEChERS ready-to-use mix)
containing 4.0
±
0.4 g MgSO
4
and 1.0
±
0.1 g NaCl. Immediately
hand-shake by inversion or by vortexing to prevent any lump
formation. Place onto a GenoGrinder shaker and shake for 1.5 min
at 1500 rpm.
Centrifuge at 4000 ×
g
at room temperature for 5 min and transfer
the supernatant (approximately 5 mL) into a 15 mL Falcon tube.
Evaporate the collected supernatant under a stream of nitrogen at
40 ± 2°C until a 0.5 mL remaining volume. A mark at the 0.5 mL
level is visible onto the tube. Do not evaporate to lower volumes to
prevent loss on evaporation.
Transfer the 0.5 mL remaining volume into a 2 mL tube and
centrifuge at 17000 ×
g
at room temperature for 5 min.
Transfer the clear supernatant into an HPLC vial for further LC-
MS/MS analysis.
(
c
)
Reagent blank
.—In order to control any contamination
during the sample workup, a reagent blank must be analyzed along
with each series of routine samples. Water is used instead of milk.
Proceeded exactly as described in
E
(
a
) and (
b
).
F. Instrumental Conditions
(
a
)
LC-MS/MS analysis
.—Where a specific instrument is cited,
an alternative may be used provided it has the same or better
characteristics. As well, an alternative HPLC column may be used
provided it allows a retention time of the eluting analyte that is at
least twice the retention time corresponding to the void volume of
the column.
(
1
)
HPLC conditions.—
Using anAgilent 1200 SL HPLC system
(
see
Table
2015.03B
).
See
Table
2015.03C
for LC gradient.
Using these conditions, the compound elutes at approximately
1.7 min (
see
Figures
2015.03B
–
E
).
(
2
)
MS parameters.
—MS parameters (Tables
2015.03D
and
E
)
are obtained by separately syringe-infusing standard solution
(approximately 1 µg/mL) of each unlabeled and labeled compounds
(syringe flow rate of 10 µL/min) along with the HPLC flow at
0.45 mL/min using a T connector. The HPLC flow is constituted
with 10% A,
C
(
i
)(
1
), and 90% B,
C
(
i
)(
2
).
(
b
)
Instrument check test
.—Before routine analysis, ensure
that the LC-MS/MS apparatus is working in conditions such as
the method remains fit for purposes. This involves to inject a low
concentration calibrant [e.g., STD 2,
C
(
h
)] to check that sensitivity
of the instrument is adequate.
G. Operating Procedure and Determination
(
a
)
Sequence setup
.—Inject solutions in the following order:
acetonitrile (as blank solvent) at least three times, standard
solutions,
C
(
h
), acetonitrile at least three times, reagent blank,
E
(
c
), extract solutions,
E
(
b
), and standard solutions,
C
(
h
), again.
Inject acetonitrile after each three to four extract solutions to check
for any carry-over.
(
b
)
Calibration
.—Draw a calibration curve by plotting peak
area ratio of the analyte and its IS (=
y
axis) against concentration
ratio of the analyte and its IS (=
x
axis). Calculate the slope and
intercept by linear regression. Check the linearity of the calibration
[regression coefficient R
2
should be higher than 0.98 and relative
standard deviation of the average of response factors (=
y/x
) should
be <15%].
(
c
)
Identification and confirmation
.—Sodium fluoroacetate is
identified and confirmed when the following criteria are fulfilled (1).
(
1
) The ratio of the chromatographic retention time of the
analyte to that of its IS, i.e., the relative retention time, corresponds
to that of the averaged relative retention time of the calibration
solutions within a ±2.5% tolerance.
Table 2015.03C. LC gradient used for analysis of sodium
fluoroacetate
Time, min
A, %
B, %
0
10
90
2.0
10
90
3.0
60
40
4.5
60
40
4.6
10
90
8.0
10
90
Table 2015.03B. HPLC conditions for the analysis of sodium
fluoroacetate
Mobile phase A
Water containing 5 mM ammonium
formate and 0.01% formic acid,
C
(
i
)(
1
)
Mobile phase B
Acetonitrile,
C
(
i
)(
2
)
Injection volume
20 µL
Column
Waters Acquity UPLC BEH Amide,
2.1
×
100 mm, 1.7 µm
Column oven temp.
45°C
Flow rate
0.45 mL/min
Needle wash
In flush port for 20 s using acetonitrile–
water (1 + 1) solution,
C
(
i
)(
3
)
Diverter valve
HPLC flow is directed into the MS detector
between 1.0 and 2.5 min
Gradient
LC gradient is described in Table
2015.03C
Candidates for 2016 Method of the Year
34