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© 2015 AOAC INTERNATIONAL
(
1
) Calculate volume of stock standard to be used
V
(µL) = 10
× 1000 × 10/
C
.
(
2
) Transfer
V
μL monofluoroacetate stock standard to a 10 mL
volumetric flask.
(
3
) Add water to the 10 mL mark and mix well.
(
h
)
Acetonitrile–water (60 + 40, v/v)
.
(
1
) Transfer 60 mL acetonitrile to 100 mL glass bottle.
(
2
) Add 40 mL water and mix well.
(
i
)
Acetonitrile–water (25 + 75, v/v)
.
(
1
) Transfer 25 mL acetonitrile to 100 mL glass bottle.
(
2
) Add 75 mL water and mix well.
(
j
)
0.1 N HCl–ethanol (50 + 50, v/v).
(
1
) Transfer 0.5 mL hydrochloric acid to 100 mL glass bottle.
(
2
) Add 50 mL water.
(
3
) Add 50 mL ethanol and mix well.
(
k
)
Pyridine, 3% in ethanol.
(
1
) Transfer 3 mL pyridine to 100 mL glass bottle.
(
2
) Add 97 mL ethanol and mix well.
(
l
)
2-NPH
.—Approximately 0.1 M in 0.1 N HCl–ethanol
(50 + 50, v/v).
(
1
) Weigh 0.375 ± 0.010 g wet powder into 25 mL volumetric
flask.
(
2
) Add ~20 mL 0.1 N HCl–ethanol (50 + 50, v/v).
(
3
) Sonicate and swirl until dissolved.
(
4
) Fill to volume with 0.1 N HCl–ethanol (50 + 50, v/v). Mix
well. Expiration 5 days.
(
m
)
EDC.
—0.25 M in 3% pyridine in ethanol.
(
1
) Weigh 1.20 ± 0.05 g EDC in weighing pan.
(
2
) Transfer to suitable storage vessel (minimum 25 mL capacity
with a wide mouth).
(
3
) Add 24 mL 3% pyridine in ethanol and mix well. Expiration
5 days.
(
n
)
Mobile phase buffer
.—200 mM ammonium acetate in water.
(
1
) Weigh 1.54 ± 0.05 g ammonium acetate in weighing pan.
(
2
) Transfer to 100 mL volumetric flask with water.
(
3
) Add 50 mL water and swirl until dissolved.
(
4
) Fill to volume with water and mix well. Expiration 5 days.
(
o
)
Mobile phase A
.—10 mM ammonium acetate in water.
(
1
) Transfer 25 mL mobile phase buffer to 500 mL mobile phase
bottle.
(
2
) Add 475 mL water and mix well.
(
3
) Sonicate for 5 min. Expiration 5 days.
(
p
)
Mobile phase B
.—10 mM ammonium acetate in acetonitrile–
water (95 + 5, v/v).
(
1
) Transfer 25 mL mobile phase buffer to 500 mL mobile phase
bottle.
(
2
) Add 475 mL acetonitrile and mix well.
(
3
) Sonicate for 5 min. Expiration 5 days.
(
q
)
Calibration standards (STD 1–6)
.
(
1
) Add volume aliquots (µL) listed in Table
2015.04A
to glass
centrifuge tubes. For method blank, use 1 mL acetonitrile–water
(60 + 40, v/v).
(
2
) Vortex.
(
3
) Perform steps
E
(
j
)–(
m
) concurrently with sample extracts.
(
4
) Transfer 250 µL of each calibration standard solution to
2 mL autosampler vial.
(
5
) Add 750 µL water and mix well. Expiration 48 h. Prepared
calibration standards contain 1, 2, 5, 10, 50, and 100 ng/mL,
respectively, of derivatized monofluoroacetate along with 10 ng/mL
each of derivatized internal standard.
E. Procedure
(
a
) Weigh 1.00 g powdered sample into 50 mL polypropylene
centrifuge tube.
(
b
) For QC overspikes add 50 µL of QC1 overspike solution
(QC Low, 25 ng/g) or 10 µL of QC2 overspike solution (QC Med,
100 ng/g) or 50 µL of QC2 overspike solution (QC High, 500 ng/g).
(
c
) Add 9 mL water.
(
d
) Shake by hand until homogenous.
(
e
) Transfer 1 mL liquid sample to a 15 mL centrifuge tube.
(
f
) Add 5 µL internal standard working solution and vortex.
(
g
) Add 1.5 mL (2 × 0.75 mL) acetonitrile and shake by hand
for 10 s.
(
h
) Centrifuge at 3000 rpm for 5 min at 5°C.
(
i
) Transfer 1 mL of supernatant to glass tube with screw cap.
(
j
) Add 0.5 mL 2-NPH reagent and vortex briefly.
(
k
) Add 0.5 mL EDC reagent and vortex briefly.
Figure 2015.04B. QC overspike at the method limit of
quantitation (10 ng/g powder).
min
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
%
0
100
MRMof 2 channels,ES-
216 > 186
03121510 Smooth(Mn,2x2)
QC1
3.198e+005
MFA-2NPH_IS
4.04
min
%
0
100
MRMof 2 channels,ES-
212 > 182
03121510 Smooth(Mn,2x2)
QC1
3.895e+004
MFA-2NPH
4.05
3.16
2.83 3.35
4.74
4.22
6.59
4.81
6.27
6.21
5.48
4.91
6.66
6.90
min
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
%
0
100
MRMof 2 channels,ES-
216 > 186
03121513 Smooth(Mn,2x2)
QC2
3.434e+005
MFA-2NPH_IS
4.04
min
%
0
100
MRMof 2 channels,ES-
212 > 182
03121513 Smooth(Mn,2x2)
QC2
9.197e+004
MFA-2NPH
4.05
3.16
2.83 3.35
4.73
6.59
4.81
6.27
5.48 5.88
6.65
Figure 2015.04C. QC overspike Low at 25 ng/g powder.
Table 2015.04D. Typical method performance indicators
achieved during in-house validation
Accuracy and
precision (
n
= 9)
10 ng/g 25 ng/g 100 ng/g 500 ng/g
Avg. accuracy, % 116
102
109
100
Precision (RSD), % 3.8
2.4
2.6
3.4
Candidates for 2016 Method of the Year
49