55 / 363
1138
Reddy
: J
ournal of
AOAC I
nternational
Vol. 98, No. 4, 2015
(
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 µLwater 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.
(
l
) Cap tightly and incubate in water bath at 80°C for 5 min.
(
m
) Cool to room temperature.
(
n
) Using a glass pipet transfer sample to 50 mL
polypropylene centrifuge tube.
(
o
) Add water to a total volume of 15 mL. Cap and invert
10 times.
(
p
) Condition Envi-Chrom P SPE cartridge (500 mg, 6 mL)
and reservoir with 10 mL ethyl acetate, 5 mL acetonitrile, and
10 mL water. Leave 1–2 mm water on the cartridge.
(
q
) Load entire sample (15 mL) and allow it to pass through
the cartridge.
(
r
) Wash cartridge with 5 mL water.
(
s
) Discard reservoirs.
(
t
) Wash cartridge with 1 mL acetonitrile.
(
u
) Dry cartridge at 5 psi vacuum for 5 min.
(
v
) Elute cartridge with 2 × 5 mL ethyl acetate and collect in
15 mL polypropylene centrifuge tube.
(
w
) Using a glass pipet remove residual aqueous layer from
bottom of tube.
(
x
) Evaporate extract to dryness under N
2
gas at 50°C.
(
y
) Reconstitute with 0.4 mL water–acetonitrile (75 + 25,
v/v).
(
z
) Vortex for 10 s, sonicate for 1 min, and vortex again for
10 s.
(
aa
) Filter through 0.2 µm PTFE syringe filter into 2 mL
centrifuge tube.
(
bb
) Using a 100 µL micropipet transfer 270 µL (3 × 90 µL)
extract to autosampler vial with insert.
F. Instrumental Conditions
See
Table
2015.04B
.
G. Data Processing
Results are read from the calibration curve and multiplied by
10 (dilution factor from powder to liquid;
see
Table
2015.04C
).
H. Method Acceptance Criteria
(
a
) Calibration curvesmust have coefficients of determination
R
2
of ≥0.99.
(
b
) Calibration curve residuals (relative error) must be ≤15%.
(
c
) Method blank cannot have detectable levels of MFA.
(
d
) QC overspike (apparent) recovery must be within
70–130% of the target value for QC Low, QC Med, and QC
High.
I. Demonstrated Method Performance
(
a
) Accuracy of overspiked samples over 3 days and at four
different levels ranged between 95–128% during qualification.
Table
2015.04D
shows the average recovery and precision at
each overspike level.
(
b
) The method detection limit (MDL) and the method
quantitation limit (MQL) for MFA in powders are 2 and 10 ng/g,
respectively.
J. Example Chromatograms
See
Figures
2015.04B–E
for example chromatograms.
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
MRMof2 channels,ES-
216 > 186
03121510 Smooth(Mn,2x2)
QC1
3.198e+005
MFA-2NPH_IS
4.04
min
%
0
100
MRMof2 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
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
Table 2015.04C. Processing method
Quantitation trace
212 > 182 Smoothing iterations
2
Internal standard trace 216 > 186 Smoothing width
2
Response type
Ratio to IS Polynomial type
Linear
Predicted RT
a
4.0 min
Origin
Excluded
RT window
±0.2 min
Weighting
1/X
a
RT = Retention time.
Candidates for 2016 Method of the Year
54