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

571

6.2 Number of Laboratories:

572 573

Based on AOAC Appendix D guidelines, studies must have a minimum of 8 laboratories submitting valid data (to avoid unduly

large confidence bands about the estimated parameters).

To minimize potential bias, no more than 25% of the laboratories with data included in the final dataset may come from the 574 same organization. For this purpose, the term “organization” includes companies (test kit manufacturers, method developers, 575 food processors, etc.), regulatory bodies, government agencies, or any other body. [Appendix M] 576 6.3 Matrix Study 577 The collaborator sites will perform the matrix studies for each claimed gluten source in at least one of the incurred matrices 578 for each matrix category claimed in the method developer study, following the rotation of claimed gluten sources shown in Tables 579 1 or 2, depending on the method claims. The selection of which matrices/surfaces/solutions are analyzed should be reflective of 580 the range of difficulty associated with the claimed matrices. 581 If the method developer study consisted of only individual matrices, rather than matrix categories, then the collaborator 582 study will test at least one incurred matrix for every 5 matrices tested in the method developer study, as shown in Table 4. The 583 collaborator sites must also analyze at least one environmental surface/CIP solution for every five claimed. If both environmental 584 surfaces and CIP solutions are claimed as matrices, and only one is to be included in the collaborative study, the environmental 585 surface should be the chosen matrix. 586

Claimed Matrices Matrices tested by Independent or Collaborator Labs

1-5

6-10

11-15

16-20

1

2

3

4

587 588 589 590

Table 4 . Number of matrices to be tested by each independent or collaborator site, as related to the number of claimed

matrices.

The selection of the specific matrices used in the collaborative studies should be reflective of the range of difficulty and

matrix category associated with the claimed matrices.

591

6.4 Test Materials:

Appendix D requires a minimum of 5 materials be used in the collaborative study. (11) Three materials are allowed but only 592 when a single specification is involved for a single matrix. For the purposes of this requirement, a test material is defined as a 593 combination of the matrix, gluten source and concentration to which the method performance parameters apply. This means 594 that, at a minimum, single gluten-source/concentration test materials, at five different concentrations, could be tested across 595 five different matrices. The use of more than 5 materials is recommended. 596 Two blind-coded replicate test portions should be analyzed by each laboratory for each test material (i.e., each matrix- 597 concentration combination). For each matrix, the concentration levels must include a blank (zero) and a level at less than or equal 598 to two times the LOQ stated in the kit insert (as long as this is less than or equal to 20 mg/kg, otherwise test at 20 mg/kg). The 599 remaining concentrations should be distributed throughout the quantification range. (7) 600 Incurred test materials are required for estimation of precision, LOD/LOQ, and recovery. See Annex B for description of best 601 practices for incurred matrix preparation. 602 6.5 Data Analysis 603 All individual data values must be reported. 604 Data analysis will be conducted according to the procedures described in Appendix D. Specifically, the following must be 605 performed and reported: 606  Outliers should be evaluated as described in Appendix D. 607  Recovery must be reported, with calculations using the known quantity of target present in incurred test materials 608 based on gravimetric calculations and accounting for any mass balance changes occurring during food processing (e.g., 609 moisture loss during baking). 610  Precision estimates reported must include both repeatability (S r and RSD r ) and reproducibility (S R and RSD R ). 611  LOD/LOQ 612