249 / 258
1130
Mottier et al.
: J
ournal of
AOAC I
nternational
Vol. 98, No. 4, 2015
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 an Agilent 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.
(
2
) The peak area ratios from the different transition reactions
recorded for the analyte and its IS are within the tolerances fixed
by the EU criteria (1) as shown in Table
2015.03E
.
(
d
)
Time of analysis
.—Following this procedure, 20 samples
can be analyzed within 24 h.
H. Calculations and Expression of Results
(
a
)
Calculation
.—Calculate the mass fraction,
w
, of sodium
fluoroacetate in microgram per kilogram of sample (µg/kg),
using the equation:
a
is
is
a
m
m
S
I
A
A
x
w
−
=
where A
a
= peak area of the analyte in the sample (transition
reaction used for quantification); A
is
= peak area of the IS in the
sample (transition reaction used for quantification); I = intercept
of the regression line for the transition reaction used for
quantification; S = slope of the regression line for the transition
reaction used for quantification; m
is
= mass of IS added to the
test portion, in ng (i.e., 10 ng for powdered sample and 50 ng for
liquid sample); m
a
= mass of the test portion, in g (i.e., 1 g for
powdered sample and 5 g for liquid sample).
(
b
)
Expression of results
.—Report the result of sodium
fluoroacetate in µg/kg with one significant figure. Nondetected
amount must be expressed as <1 µg/kg.
I. Performance Characteristics
The method was validated using samples provided by
the AOAC Stakeholder Panel on Infant Formula and Adult
Nutritionals (SPIFAN). Infant formulae considered are
described in Table
2015.03F
.
Validation was performed according to the protocol described
in SANCO/12571/2013 (2): precision data were obtained by
spiking each sample at 1 and 10 µg/kg concentration levels
(respectively corresponding to 1 × LOQ and 10 × LOQ level).
At least two operators were involved in these experiments,
each performing five replicates at the mentioned fortification
levels on two different days (leading to a total of 10 separate
experiments for each fortification level). Nonfortified samples
were analyzed as well to verify absence of the pesticide before
fortification trials.
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
249