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

ANNEX B

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PREPARATION OF MATERIALS FOR GLUTEN METHOD VALIDATION 2 Until such time as a reference materials are available, the gluten source for all prepared samples should 3 be commercial, unbleached whole wheat, whole rye, whole barley or whole oat flour. 4 The chosen flour should be analyzed for Dumas or Kjeldahl nitrogen. Convert to percent crude protein by 5 multiplying the nitrogen value by 5.83. Then convert to percent gluten by multiplying the crude protein 6 value by the following factors, depending on the grain: 7 • Wheat 0.74 8 • Rye 0.52 9 • Barley 0.78 10 • Oats 0.15 11 These conversion factors are suggestions, and may vary across different grain samples. The factors come 12 from two publications (1,2); the conversion factors for wheat rye and barley are based on the wet 13 chemistry method described in Wehling and Scherf (2). Method developers may also use the wet chemical 14 method in Wehling and Scherf (2) to arrive at the gluten content for their own wheat, rye and barley 15 flours. 16 (1) Schalk K, Lexhaller B, Koehler P, Scherf KA (2017) Isolation and characterization of gluten protein types from wheat, rye, barley 17 and oats for use as reference materials. PLoS ONE 12(2): e0172819. doi:10.1371/journal.pone.0172819 18 (2) Wehling, P and Scherf, KA (2020) Preparation of Validation Materials for Estimating Gluten Recovery by ELISA According to 19 SMPR 2017.21. Journal of AOAC International 103(1): 210-215. 20 21 Finally, convert the percent gluten to mg/kg (ppm) gluten by multiplying the result by 10,000. 22 As an example, a barley flour is tested and found to have a Dumas nitrogen level of 1.5%. This is multiplied 23 by 5.83 to attain a crude protein level of 8.75%. Using the conversion factor for barley, the 8.75% crude 24 protein is multiplied by 0.75 to obtain the gluten percent of 6.56%. This percent value is then multiplied 25 by 10,000 to estimate the mg/kg (ppm) value at 65,600. This is equivalent to 65.6 mg of gluten per gram 26 of flour. 27 Making Spiked Materials 28 Bulk spiked materials may be prepared for the selectivity, stability and lot-to-lot studies, and bulk spikes 29 of raw materials are often made prior to the processing steps when making incurred samples. 30 These methods can be used for any material that has a small particle size or uniform consistency, including 31 flours, baking mixes, spices, meats, sauces, dressings, ice cream (melted), etc. They can also be used in 32 other matrices that can be dried and ground to a flour-like consistency, such as nuts, seeds, and bread 33 crumbs. 34 Thorough blending is key to a successful trial. For dry materials like flours, or for liquid consistencies, 35 blending can be done in a blender or tumbler-style mixer, or even by manual tumbling of material in a 36 zippered plastic bag. Add the spike material uniformly within the matrix, rather than adding it all in one 37 location prior to blending it in. Making spikes in very fine matrices with small particle size can be difficult, 38