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1138  Reddy : J ournal of AOAC I nternational Vol. 98, No. 4, 2015

( 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.

Table 2015.04C. Processing method Quantitation trace

212 > 182 Smoothing iterations

2

Internal standard trace 216 > 186 Smoothing width

2

Response type Predicted RT a

Ratio to IS Polynomial type

Linear

4.0 min

Origin

Excluded

RT window

±0.2 min

Weighting

1/X

a  RT = Retention time.

( 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.

03121510 Smooth(Mn,2x2) QC1

MRMof2 channels,ES- 212 > 182

MFA-2NPH 4.05

3.895e+004

100

4.74

6.59

6.66

4.81

%

6.27

6.90

3.16 2.83 3.35

6.21 5.48 4.91

4.22

( t ) Wash cartridge with 1 mL acetonitrile. ( u ) Dry cartridge at 5 psi vacuum for 5 min.

0

min

03121510 Smooth(Mn,2x2) QC1

MRMof2 channels,ES- 216 > 186

MFA-2NPH_IS 4.04

3.198e+005

100

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

%

0

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

Figure 2015.04B. QC overspike at the method limit of quantitation (10 ng/g powder).