AOAC Gluten Qualitative Validation Guidance-FINAL (July 2023)

Annex B

Making Incurred Materials The section above, Making Spiked Materials, describes the initial steps in making an incurred material. The spiking must occur prior to the major processing step in order for the end product to be considered an incurred matrix. Further considerations for common types of processing are provided below Baked, Fried or Dehydrated Materials Baking, frying and dehydrating are processing methods that can be reasonably replicated at a small scale, in a laboratory. The same process applies for each. When possible, weigh the incurred material before and after processing. Any change in the analyte concentration above or below the expected value should be accounted for by the change in mass. When exact ppm values are needed, for example for a quantitative method, the moisture/weight change from processing must be accounted for in determining the amount of spike material to be added. If the entirety of the material cannot be weighed before and after processing, additional analyses can be performed to determine the potential analyte gain or loss, such as moisture content, protein, or zinc/other metals. If moisture/weight change results in a slightly higher ppm value than intended, higher level incurred test materials can be mixed with blank, processed material to achieve various concentrations. The lowest concentration achieved in this way should not be less than 10% of the concentration of the high-level incurred material. Larger discrepancies require a second incurred matrix to be made at a lower level. Pressure Treated/High Heat/Extruded These are processes that cannot normally be replicated outside of a manufacturing facility. If a manufacturer is particularly interested in the development of the assay, the kit developer may be able to partner with them to make gluten spikes on a pilot scale, using a similar method as described above for baked, fried and dehydrated products. In the absence of access to a manufacturing plant, some highly processed matrices can be incurred through “fortification”. An example would be a whole wheat puffed/extruded breakfast cereal. A pilot plant could create a mid-level spike (100 ppm, for example), which could be diluted down in a similarly processed blank matrix to create lower concentrations. Any validated method can be used to verify the absence of gluten in the non-gluten-containing matrix. Making Environmental Surface Test Materials Determine the surface area that’s expected to be swabbed. Typical area is 25 cm 2 – 100 cm 2 (approx. 4 in. 2 - 16 in. 2 ) Make suspensions from the flour in the kit extraction solution, or 60% ethanol solution. Create solutions at gluten concentrations ( µ g/ml) around the expected sensitivity level of the method, as described in the validation requirements.