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© 2015 AOAC INTERNATIONAL
(
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 curves must 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.
K. Example Calibration Curve
See
Figure
2015.04F.
References: (1)
J. Chromatogr. A
881
, 365(2000)
(2)
J. Chromatogr. A
1139
, 271(2007)
J. AOAC Int . 98 , 1135(2015)DOI: 10.5740/jaoac.int.2015.04
AOAC SMPR 2015.001
J. AOAC Int. 98 , 1092(2015)DOI: 10.5740/jaoac.int.SMPR2015.001
Posted: October 15, 2015
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
03121519 Smooth(Mn,2x2)
QC4
2.967e+005
MFA-2NPH_IS
4.04
min
%
0
100
MRMof 2 channels,ES-
212 > 182
03121519 Smooth(Mn,2x2)
QC4
1.622e+006
MFA-2NPH
4.05
Figure 2015.04E. QC overspike High at 500 ng/g powder.
Figure 2015.04F. Calibration curve and residual plot.
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
03121516 Smooth(Mn,2x2)
QC3
3.304e+005
MFA-2NPH_IS
4.04
min
%
0
100
MRMof 2 channels,ES-
212 > 182
03121516 Smooth(Mn,2x2)
QC3
3.995e+005
MFA-2NPH
4.05
4.81
6.58
6.27
Figure 2015.04D. QC overspike Med at 100 ng/g powder.
Candidates for 2016 Method of the Year
50