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

When more than four factors are varied, a full-factorial experiment can become prohibitively large. 355 When varying five or more, a fractional factorial design, or “screening” design, can be used. A limitation 356 of these designs is that some of the interaction effects may be confounded with each other. But if a 357 resolution IV or V design is used, none of the main effects will be confounded with each other. The 358 following are examples of 2 5-1 and 2 6-2 designs, for five and six factors respectively. The 2 5-1 design is a 359 resolution V, and the 2 6-2 design is a resolution IV, meaning that main effects are at most confounded 360 with 3rd-order or higher interactions.

361 362

Varying 5 Factors in a 2 5-1 Design, resolution V

Sample Size

Extract. Time 20 min 20 min 40 min 40 min 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

Extract Temp

Sample Load

Conjugate Dilution

Run

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

45 C 45 C 45 C 45 C 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

1 2 3 4 5 6 7 8 9

1:8

50 µ l 50 µ l 50 µ l 50 µ l 50 µ l 50 µ l 50 µ l 50 µ l

1:12 1:12

1:8

1:12

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

363 364 365 366 367 368 369 370 371 372 373 374

Table 4.

Varying 6 Factors in a 2 6-2 Design, resolution IV

Sample Size

Extract. Time 20 min 20 min 40 min 40 min

Extract Temp

Sample Load

Conjugate Dilution

TMB Time

Run

1.5g 2.5g 1.5g 2.5g

45 C 45 C 45 C 45 C

15 min 25 min 25 min 15 min

1 2 3 4

1:8

50 µ l 50 µ l 50 µ l 50 µ l

1:12 1:12

1:8