AOAC Gluten Quantitative Validation Guidance-Round 1(Nov 2023)

1.5g 2.5g 1.5g 2.5g 1.5g 2.5g

20 min 20 min 40 min 40 min 20 min 20 min 40 min 40 min 20 min 20 min 40 min 40 min 30 min

75 C 75 C 75 C 75 C 45 C 45 C 45 C 45 C 75 C 75 C 75 C 75 C 60 C

15 min 25 min 25 min 15 min 25 min 15 min 15 min 25 min 25 min 15 min 15 min 25 min 20 min

5 6 7 8 9

1:12

50 µ l 50 µ l 50 µ l 50 µ l

1:8 1:8

1:12 1:12

150 µ l 150 µ l 150 µ l 150 µ l 150 µ l 150 µ l 150 µ l 150 µ l 100 ml

10 11 12 13 14 15 16

1:8 1:8

150 µ l 150 µ l 150 µ l 150 µ l 150 µ l 150 µ l

1:12

1:8

1:12 1:12

1:8

N

2g

1:10

Table 5. 375 Additional fractional factorial designs can be generated in R – see the instructions later in this 376 document.

377 378

Plackett-Burman Designs 379 When more than six factors are varied, a Plackett-Burman design may be needed. This type of design 380 reduces the total number of experimental runs while still allowing for the analysis of the main effects of 381 individual factors. A concern with Plackett-Burman designs is that the main effects of the individual 382 factors are confounded with interaction effects, so it can’t be determined if any significant effects are 383 due to changes in an individual factor, or to that plus the changes in another factor. If it is important to 384 discriminate between the individual effects and interaction effects, then the few significant factors 385 identified by the Plackett-Burman design may be used in a separate full factorial experiment. 386 Examples of designs where seven or eight factors are varied are given below (our hypothetical method 387 only had six factors to vary, so factors seven and eight are unnamed). With this same 12-row design you 388 may test up to 11 factors – examples of this can be found in the NIST Engineering Statistics Handbook. 389 The row labeled “N” at the bottom of each table describes the “normal” parameter values for the assay, 390 but this is only included for informational purposes and does not need to be tested as part of the 391 experiment.

392 393 394 395 396

Varying 7 Parameters in a Plackett-Burman Design

Run

Sample Size

Extract. Time 40 min 40 min 20 min 20 min 40 min 20 min 20 min 20 min 40 min

Extract Temp

Sample Load 150 µ l 150 µ l 150 µ l 50 µ l 50 µ l 50 µ l 50 µ l 150 µ l 50 µ l

Conjugate Dilution

TMB Time

Factor 7

1 2 3 4 5 6 7 8 9

2.5g 1.5g 1.5g 2.5g 1.5g 1.5g 1.5g 2.5g 2.5g

75 C 45 C 75 C 45 C 45 C 75 C 45 C 45 C 45 C

25 min 25 min 25 min 25 min 15 min 25 min 15 min 15 min 25 min

High Low High High High Low High Low Low

1:8 1:8 1:8

1:12

1:8

1:12 1:12

1:8

1:12