AOACRIGlutenMethods-2017Awards

1352  Lacorn & Weiss : J ournal of AOAC I nternational Vol. 98, No. 5, 2015

Table 2. Gluten concentrations determined by R5 competitive ELISA after eliminating laboratories E, F, and K Gluten concentration, mg/kg a 1 b 2 3 4 5 6 7 Repeat Lab 1 2 1 2 1 2 1 2 1 2 1 2 1 2 A 2.13 5.80 23.6 20.5 111.6 93.9 4.47 7.73 7.6 8.62 46.7 47.2 153.0 170.0 B 1.46 2.66 40.8 13.8 151.4 127.4 2.98 2.13 10.6 5.1 38.8 53.0 163.6 122.8 C 5.30 10.6 34.2 c 82.2 c 192.2 107.6 6.12 1.90 12.8 12.6 47.2 67.4 181.4 143.4 D 0.74 1.77 23.8 28.6 175.2 97.6 –3.35 –3.41 9.8 11.0 33.0 60.2 106.4 107.6 G 6.06 4.30 32.4 32.0 216.2 d 208.2 d 3.29 –2.34 15.0 14.0 46.8 85.4 192.8 203.0 H 7.02 1.56 44.4 26.2 145.6 32.8 5.79 3.17 20.5 16.1 38.8 31.1 94.6 88.9 I –0.65 –1.33 22.2 13.8 101.2 64.4 –0.89 –0.62 5.4 4.2 35.8 45.0 118.4 75.0 J –1.50 1.14 21.2 20.0 121.8 128.8 –0.73 –1.63 7.4 8.0 45.6 58.3 132.9 139.2 L 1.69 –0.33 39.8 49.0 224.8 d 228.8 d –1.83 3.39 13.2 11.6 64.0 67.2 171.6 244.6 M –0.66 4.13 19.9 19.3 129.4 133.6 –2.27 –0.62 10.0 8.6 36.1 39.6 161.7 120.4 N 0.04 0.76 34.2 18.4 97.0 108.6 1.84 4.41 10.8 9.2 43.4 44.6 117.6 154.4 O 1.57 0.41 19.1 16.5 110.7 136.6 –0.11 1.26 11.7 8.2 51.3 46.3 152.8 164.6 P 5.96 0.84 25.4 24.8 149.4 111.2 1.54 1.33 12.6 10.8 38.2 46.2 194.8 111.2 a  The calculation of the concentrations of the gluten-containing samples 2, 3, 5, 6, and 7 was done on the basis of a cubic spline function using the RIDA ® SOFT Win software; the statistics of the gluten-free samples 1 and 4 were calculated on the basis of a second-order polynomial function; values for blinded samples are given as repeat 1 or repeat 2. b  For samples 1–7 see Table 1. c  Means outlier according to the Cochran test. d  Means outlier according to the double Grubbs’ test.

the possibility of gluten contamination in the laboratory and incorrect pipetting. As a result of these deviations, all data from Laboratories E, F, and K were excluded from the statistical evaluation. For sample 5 (naturally contaminated syrup), all values were calculated by cubic spline. Due to the fact that some OD values were below the OD values of standard 2 (10 ng/mL prolamin; corresponds to concentration of 10 mg/kg in the sample), these values were extrapolated by the software. For the gluten-free samples 1 and 4 the RIDA ® SOFT Win software returned only a result of <10 mg/kg, and extrapolation led to unrealistic values. To be able to use the results of the analysis of the gluten- free samples 1 and 4 in the performance statistics, estimates of concentration values for these samples were required. For this purpose, the calibration curves were constructed by using a second-order polynomial model and used to recalculate the results for samples 1 and 4 (7). This calibration provided an estimate of concentrations for the gluten-free samples (Tables 1 and 2). The remaining data of 13 laboratories are shown in Table 2 and were used to calculate the necessary statistics. Only three outlying values were identified according to AOAC INTERNATIONAL guidelines (12). These are indicated in Table 2 by the superscripts “c” (for a Cochran outlier) and “d” (for a double Grubbs’ outlier). The performance statistics without outliers are shown in Table 2015.05. From the measured overall mean concentrations of the gluten-containing samples, recovery rates were calculated. Statistical Analysis and Discussion

J. Criteria for Acceptance of the Standard Curve The shape of the standard curve is shown in the quality assurance certificate enclosed in the test kit. Absorbances may vary between different runs (e.g., due to different temperatures or analysts). However, the shape of the standard curve should be similar to the one given in the quality assurance certificate. Minimum requirements are as follows: ( 1 ) OD at 450 nm for standard 1 higher than 0.8. ( 2 ) OD values for standards should continuously decrease with higher concentrations, especially when comparing standard 1 (0 ng/mL) and standard 2 (20 ng/mL). ( 3 ) An OD value for standard 1 that is much higher than the OD value stated in the certificate could be an indication of errors during pipetting or incubation. Collaborative Study Results After finishing the analysis, each participant sent the data to the Study Coordinator. These results are given in Table 1. After statistical analysis of the data set, three problem laboratories were identified. Further review found Laboratory F did not run the calibrators in duplicate determinations as directed. Laboratory E found no difference between calibration standards S1 and S2, and as a consequence, a high OD difference between standards S4 and S5 led to an unusual curve shape. An interview with Laboratory E also revealed technical problems during sample preparation. Laboratory K had a variation in the calibration curve that was too high, and an interview revealed Results and Discussion

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