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chneider
&
A
ndersen
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J
ournal of
AOAC I
nternational
V
ol
.
98, N
o
. 3, 2015
661
and LCV-D6). To prepare each of these solutions, combine
1 mL of each individual stock solution required for the mixture
and dilute to 100 mL final volume with acetonitrile. These
solutions are stored in glass at –20°C and protected from light
(stated stability = 1 month).
(
c
)
Working standard solutions
.—Prepare five to six working
standard solutions. Six will be described here (WS 1–6). These
are prepared by diluting aliquots (0, 50, 100, 200, 500, and
1000 µL, respectively) of the mixed intermediate standard
solution of analytes to a final volume of 10 mL with acetonitrile.
The resultant working standard solutions thus contain 0, 5, 10,
20, 50, and 100 µg/L, respectively, of the analytes. Prepare
a working standard solution of internal standards by taking
400 µL of the mixed intermediate internal standard solution
and diluting to a final volume of 10 mL with acetonitrile (final
concentration 40 μg/L). Prepare all working standard solutions
daily. These solutions may be kept at room temperature but
protected from light.
F. Sample Preparation
(
a
)
Homogenization of samples
.—Homogenize muscle
tissue with dry ice in a food processor to produce a finely
ground powder. Allow the dry ice to sublime at –20°C and then
store the homogenized tissues at –80°C. Homogenize salmon
with attached skin, but for catfish, homogenize only the skinless
filet. Remove shells, legs, and heads from shrimp prior to
homogenization.
(
b
)
Extracted matrix calibrant samples.—
Accurately weigh
2.00 g (±0.02 g) portions of homogenized negative control
tissue into each of six 50 mL disposable centrifuge tubes. Once
thawed, fortify these samples (extracted calibrants 1–6) with
100 µL aliquots of WS1, WS2, WS3, WS4, WS5, and WS6,
respectively. To each tube then add 100 μL internal standard
working solution. The extracted matrix calibrant samples
are thus fortified with 0, 0.25, 0.5, 1.0, 2.5, and 5.0 µg/kg of
analytes and 2.0 µg/kg of internal standards. Allow calibrants
to equilibrate 15 min protected from light before beginning the
extraction with the addition of hydroxylamine solution. [
Note
:
Method
2012.25
specifies that five extracted matrix calibrants
are prepared in the range 0 to 2 µg/kg with concentrations 0,
0.5, 1.0, 1.5, and 2.0 µg/kg of analytes (8). For the collaborative
study, the range was extended from 0 to 5 µg/kg to ensure
that residues found in incurred samples would fall within the
calibration range.]
(
c
)
Extraction of samples
.—Accurately weigh
2.00 g
(±0.02 g) portions of homogenized tissue into 50 mL disposable
centrifuge tubes and let thaw. Fortify thawed tissue with
100 μL internal standard working solution (2.0 µg/kg), and
allow samples to equilibrate for 15 min while protected from
light. Add hydroxylamine solution (9.5 g/L, 500 μL) to the
samples, vortex mix briefly, and allow samples to stand in
the dark for 10 min. Add acetonitrile (8 mL) and 1.0 g (±0.1)
anhydrous magnesium sulfate to each tube. Vortex mix tubes
(1 min, maximum speed), then shake tubes (10 min) using a
rotary stirrer or a multitube vortexer. Centrifuge the tubes
(2000 ×
g
, 5 min, 4°C), and transfer all supernatant to a clean
tube for evaporation. Evaporate the supernatant to dryness
(50°C, N
2
). For the salmon matrix, the point of dryness may
be a viscous oil. Reconstitute the extracted matrix calibrant
samples and test samples with 800 μL Reconstitution Solution.
Vortex mix all samples sufficiently to break up dried extracts;
for example, vortex mixing on high speed for 30 s followed
by 10 min of mixing on a multitube vortexer ensures complete
dissolution of analytes and internal standards. Transfer extracts
to microcentrifuge tubes, centrifuge at 20 000 ×
g
for 5 min, and
filter (PVDF, 0.45 μm) into autosampler vials for LC-MS/MS
analysis. The extraction results in a 2.5X concentration factor;
therefore, a calibrant or sample fortified at 1.0 µg/kg in the
seafood matrix will produce an extract with an equivalent
concentration of 2.5 µg/L in the LC vial.
G. LC-MS/MS Analysis
(
a
)
LC
.—A Waters Corp. Symmetry C
18
or comparable
column is used, with or without a guard column. The mobile
phase was made up of ammonium formate buffer (A, 0.05 M,
pH 4.5) and acetonitrile (B). The gradient program is described
in Table 1. The flow rate is 250 μL/min, the injection volume is
20 μL, and the column oven is set to 30°C. Potential carryover,
particularly from CV can be reduced by injection of water
between each test sample.
(
b
)
Triple quadrupole MS
.—A Waters Corp. Quattro LCZ
triple quadrupole, or comparable instrument is used. The
mass spectrometer is operated in the positive ion mode using
electrospray ionization. Two SRM transitions are collected
for each analyte and one SRM transition is collected for each
internal standard; these transitions are detailed in Table 2,
along with instrument parameters for the Waters Quattro LCZ
system. Conditions are optimized so that all SRM transitions
for the lowest concentration solvent calibrant are present with
an acceptable S/N (≥3).
H. Screening
The method can be used to screen test samples against a single
calibrant or to quantify samples using a full calibration curve.
A screen is accomplished by extracting test samples along with
a negative matrix control sample and a matrix sample fortified
at 0.5 µg/kg. Concentration of the test sample is estimated
by comparison of the quantification ion peak area ratio of
sample:internal standard with the corresponding ratio for the
fortified matrix sample. To confirm suspected positive samples,
test samples should be extracted and analyzed in duplicate
along with a range of fortified calibrants (including negative
control). Test samples are then quantified using the calibration
curve according to the quantification method described below
in Section
I
(8).
Table 1. LC elution gradient
Time,
min
A, %
(ammonium formate buffer)
B, %
(acetonitrile)
0
60
40
1
10
90
15
10
90
16
60
40
20
60
40
Candidates for 2016 Method of the Year
288