AOAC Gluten Qualitative Validation Guidance-FINAL (July 2023)

Q UALITATIVE G LUTEN M ETHOD V ALIDATION G UIDELINES PG . 6

gluten from each claimed gluten source at 20 mg/kg (or less, depending on the regulatory threshold of the target market/country), or two times the environmental surface detection limit for methods limited to environmental surface testing. Test material preparation is described in Annex B. The prepared test materials/surfaces will be analyzed with the candidate method as it is designed. Analyze one test portion per test material/surface. For each test portion/surface, the method should produce a positive result. In the event that the single test portion tests negative, it may be retested in 20 test portions, with no failures allowed, to rule out interference. Findings that certain matrices interfere with gluten detection should be investigated further, using additional similar matrices, to determine the full scope of interference.. Any interfering matrices must be reported in the method instructions. 4.1.3 Matrix / POD study The purpose of the laboratory matrix study is to measure the probability of detection at the claimed CDC(s), and below and above this level(s), in a controlled laboratory setting for all gluten sources, matrices and surfaces claimed in the method’s intended use statement. A matrix/POD study must be performed in each claimed matrix. In order to ensure that each claimed gluten source is represented, the gluten sources must be rotated across the claimed matrices as shown in Tables 1 and 2. The single matrix in which all gluten sources are tested, listed in Tables 1 and 2 as Matrix 1, should be the most highly processed matrix used in the validation study.

Number of Matrices Claimed

1

2

3

4

5

Wheat Barley Rye Oats

Wheat Barley Rye Oats Wheat

Wheat Barley Rye Oats Wheat

Wheat Barley Rye Oats Wheat

Wheat Barley Rye Oats Wheat

Matrix A Matrix B Matrix C Matrix D Matrix E

Barley

Barley

Barley

Rye

Rye

Oats Table 2. Rotation of gluten sources across claimed matrices for methods claiming to detect wheat, rye, barley and oats. The rotation of single gluten sources would continue for six matrices and greater. Alternatively, a matrix/POD study for a matrix category may be performed by testing each claimed gluten source, per the rotation shown in Table 1 or 2, in at least 5 examples from the category, equally distributed across each available type of processing (Annex C). Test materialss under each type of processing must be incurred. As an example, a method wishing to make a claim for the “Cereals (Not Fermented, Hydrolyzed or Fractionated)” category would need to test one matrix from each of the five provided processing categories, and in each instance, gluten would need to be added to the matrix prior to the described processing step. If a method developer was unable to access suitable equipment for preparing incurred test materials in the Pressure/Extruded type of processing, but was able to make incurred test materials for all other types of processing, they could not claim the “Cereals (Not Fermented, Hydrolyzed or Fractionated)” category. However, they could make a limited claim for “Raw, Processed, Baked, Fried and Dehydrated Cereals”. Method developers with the ability to produce fermented, hydrolyzed or fractionated matrix test materials that were incurred with gluten prior to these processes may make individual claims based on the fermentation organism, hydrolyzing agent or fractionation process. Example claims would be “Soy Tempeh fermented with Rhizopus oligosporus ”, “Modified corn starch hydrolyzed with sodium hydroxide”, or “Wheat starch fractioned with water”. For each gluten source-matrix combination, the developer must analyze a blank (no gluten) test material, one test material per each CDC, one test material at half of the lowest CDC, and one test material at least 1.5 times the highest CDC, prepared as described in Annex B. If the test material at half the lowest CDC produces a POD of 0%, an

Number of Matrices Claimed

1

2

3

4

5

Wheat Barley Rye

Wheat Barley Rye Wheat

Wheat Barley Rye Wheat

Wheat Barley Rye

Wheat Barley Rye

Matrix A Matrix B Matrix C Matrix D Matrix E

Wheat Wheat

Barley

Barley

Barley

Rye

Rye

Wheat

Table 1. Rotation of gluten sources across claimed matrices for methods claiming to detect wheat, rye and barley. The rotation of single gluten sources would continue for six matrices and greater.