AOAC 2018 Methods

Table 2018.15A. Performance characteristics using data from four-parameter logistic regression analysis for curve fitting a Samples 1–10 b Samples 11–21 c 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Labs, n 19 18 17 15 18 19 16 19 19 19 19 19 19 19 17 19 19 19 19 19 18 Replicates, n 38 36 34 30 36 38 32 38 38 38 38 38 38 38 34 38 38 38 38 38 36 Mean, mg/kg 10.8 13.7 11.0 0.9 62.1 33.4 4.0 10.0 32.8 47.9 3.1 21.0 15.9 27.2 2.1 6.3 12.9 22.0 7.2 13.5 20.3 s r , mg/kg d 2.29 1.88 1.40 0.96 5.31 4.62 0.79 9.68 5.88 7.12 1.51 5.91 3.87 5.80 2.23 2.70 5.34 5.99 1.93 2.86 2.22 s R , mg/kg e 2.29 2.05 1.96 0.96 7.35 6.82 2.52 9.99 7.27 7.96 2.20 6.15 4.68 6.88 2.23 3.44 5.79 6.75 2.31 2.92 3.67 RSD r , % f 21.1 13.7 12.7 103.9 8.5 13.8 19.8 96.7 17.9 14.9 48.4 28.2 24.3 21.3 107.2 42.6 41.3 27.2 26.6 21.2 10.9 RSD R , % 21.1 15.0 17.8 103.9 11.8 20.4 63.0 99.8 22.2 16.6 70.5 29.3 29.4 25.3 107.2 54.3 44.7 30.6 31.9 21.7 18.1 a  For raw data from each participant, including outlier calculation, refer to Annex A Tables A3 and A4 (in J. AOAC Int . manuscript). b  Samples 1–10 are as follows: ( 1 ) wheat flour in oat flour, 10 mg/kg gluten; ( 2 ) rye flour in oat flour, 10 mg/kg gluten; ( 3 ) barley flour in oat flour, 10 mg/kg gluten; ( 4 ) oat flour, naturally contaminated approximately at 2 mg/kg gluten; ( 5 ) wheat flour contaminated corn-based processed snack, 82 mg/kg gluten; ( 6 ) mixture of corn-based processed snacks, approximately 41 mg/kg gluten; ( 7 ) rice flour; contaminated at a very low gluten level; ( 8 ) flaked oats blank; ( 9 ) flaked oats low; and ( 10 ) flaked oats high. c  Samples 11–21 are as follows: ( 11 ) cereals-blank, ( 12 ) cereals-low, ( 13 ) cereals-medium, ( 14 ) cereals-high, ( 15 ) flour-blank, ( 16 ) flour-low, ( 17 ) flour-medium, ( 18 ) flour-high, ( 19 ) groats-blank, ( 20 ) groats-low, and ( 21 ) groats-high. d  s r = Precision of repeatability. e  s R = Precision of reproducibility. f  RSD r = Repeatability relative SD.

( c ) Remove samples from water bath and shake for 1 h upside- down or by a rotator at room temperature (20–25°C/68–77°F). ( d ) Centrifuge for 10 min at least 2500  g (alternatively, 2 mL of the extract can be centrifuged with high speed for 10 min in reaction caps by using a microcentrifuge). Afterward, filter the supernatant (with fluted paper filter). ( e ) This extract can be stored at room temperature for at least 7 days. ( f ) Dilute the sample 1:25 with buffer, D(f) , e.g., 40 μL extract + 960 μL buffer (1:1000 final sample dilution). Use diluted supernatant immediately in the assay within 30 min (use 100 μL per well in the assay). ( g ) If further dilution is required, a solution consisting of 1% Cocktail (patented), D(h) , 3% of 80% ethanol, G(b) , and

96% buffer, D(f) , e.g., 50 μL Cocktail (patented), 150 μL 80% ethanol, and 4800 μL buffer, should be used. Do not use the diluted samples that were already measured for further dilution because the diluted samples are stable for 30 min only. Restart the dilution using the extract obtained after filtration. G. Preparation of Components ( a )  Washing buffer .—Provided as a 10-fold concentrate, D ( g ). Before use, the buffer has to be diluted 1:10 (1+9) with distilled water (i.e., 100 mL buffer concentrate + 900 mL distilled water). Prior to dilution, dissolve possibly formed crystals by incubating the buffer in a water bath at 37°C (99°F). The diluted buffer is stable at 20–25°C (68–77°F) for 4 weeks.

Table 2018.15B. Performance characteristics using data from quadratic curve fitting a Samples 1–10 b

Samples 11–21 c

1 2 3 4 5 6 7

8 9 10 11 12 13 14 15 16 17 18 19 20 21

Labs, n 19 18 18 18 18 19 16 19 19 19 19 19 19 19 17 19 19 19 19 19 17 38 36 36 36 36 38 32 38 38 38 38 38 38 38 34 38 38 38 38 38 34 Mean, mg/kg 9.9 13.2 10.4 1.1 63.6 35.2 3.5 9.1 34.2 50.0 2.5 21.2 15.8 28.1 1.7 5.5 12.4 22.6 6.4 13.0 19.7 s r , mg/kg d 2.43 2.12 1.74 1.19 4.40 5.32 0.70 9.26 7.03 7.19 1.37 6.44 4.46 6.47 1.99 2.92 5.76 6.64 1.90 3.09 1.98 s R , mg/kg e 2.43 2.22 2.34 1.21 6.35 8.02 2.45 9.53 8.31 7.93 1.74 6.92 5.52 7.80 1.99 3.39 6.59 7.78 2.31 3.39 3.15 RSD r , % f 24.5 16.1 16.7 111.1 6.9 15.1 20.0 102.2 20.5 14.4 54.5 30.4 28.3 23.0 115.3 52.6 46.6 29.4 29.9 23.8 10.1 RSD R , % 24.5 16.9 22.4 113.6 10.0 22.8 70.4 105.2 24.3 15.8 69.1 32.7 35.0 27.8 115.3 61.0 53.3 34.4 36.3 26.2 16.0 a  For raw data from each participant, including outlier calculation, refer to Annex A Tables A3 and A4 (in J. AOAC Int . manuscript). b  Samples 1–10 are as follows: ( 1 ) wheat flour in oat flour, 10 mg/kg gluten; ( 2 ) rye flour in oat flour, 10 mg/kg gluten; ( 3 ) barley flour in oat flour, 10 mg/kg gluten; ( 4 ) oat flour, naturally contaminated approximately at 2 mg/kg gluten; ( 5 ) wheat flour contaminated corn-based processed snack, 82 mg/kg gluten; ( 6 ) mixture of corn-based processed snacks, approximately 41 mg/kg gluten; ( 7 ) rice flour; contaminated at a very low gluten level; ( 8 ) flaked oats blank; ( 9 ) flaked oats low; and ( 10 ) flaked oats high. c  Samples 11–21 are as follows: ( 11 ) cereals-blank, ( 12 ) cereals-low, ( 13 ) cereals-medium, ( 14 ) cereals-high, ( 15 ) flour-blank, ( 16 ) flour-low, ( 17 ) flour-medium, ( 18 ) flour-high, ( 19 ) groats-blank, ( 20 ) groats-low, and ( 21 ) groats-high. d  s r = Precision of repeatability. e  s R = Precision of reproducibility. f  RSD r = Repeatability relative SD. Replicates, n

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