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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.

257