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Background: In September 2017, the AOAC INTERNATIONAL Stakeholder Panel for Alternative Methods adopted Standard Method Performance Requirement (SMPR®) 2017.021, “Quantitation of Wheat, Rye, and Barley Gluten in Oats,” as guidance for the validation of methods for measuring gluten in oat products. The SMPR requires prospective methods to demonstrate adequate recovery (50–200%) based on the analysis of a set of reference samples. Objective: This document provides specific methods and data on the preparation of such validation materials and their analysis by an R5 ELISA kit to demonstrate the SMPR recovery estimation procedure. Methods: Seven reference samples were made by spiking wheat, rye, and barley into gluten-free oat flour at two levels, 10 and 20 mg/kg. The levels of gluten were determined by a wet chemical method based on the Codex Alimentarius definition of gluten. Results: The recoveries for wheat, rye, and barley were 122, 425, and 349%, respectively, for the R5 ELISA kit. The wet chemical method for estimating gluten in a sample of pure grain demonstrated repeatability relative SDs ranging from 1.40 to 2.75%. Conclusions: The reference materials are suitable to estimate ELISA kit responses to wheat, rye, and barley and calculate recoveries. Highlights: A series of oat flours spiked with wheat, rye, and barley flours were developed to be used as reference materials. A wet chemical method was established to estimate gluten contents based on the Codex definition. The reference materials are available for purchase to support further method development and validation.

Preparation of Validation Materials for Estimating Gluten Recovery by ELISA According to SMPR 2017.021