Gluten Analysis and Regulation: Scientific, Analytical, and Economic Dimensions of Gluten-Free Assurance.

Gluten-free regulations require analytical systems that can reliably measure gluten in wheat, rye, and barley, and, in some jurisdictions, in oats across diverse food ingredients and processed foods. Examples include baked goods, fermented products such as beer, and extruded snack foods, where processing can alter gluten extractability and antibody recognition. This review examines the scientific, analytical, and regulatory factors that determine whether gluten quantification can support robust enforcement of the 20 mg gluten/kg threshold used in most jurisdictions, with a focus on method performance criteria (MPCs). The molecular complexity of gluten and the structural changes induced by baking, fermentation, and other processing steps influence extractability and epitope availability. These effects contribute to variable results across enzyme-linked immunosorbent assays that use different antibodies, extraction chemistries, and calibration standards. The widespread use of PWG gliadin (gliadin reference material) as a calibrant supports harmonized calibration for wheat-based analyses. Still, it can introduce bias because it does not reflect the gluten composition in processed or mixed-cereal foods. Improved incurred reference materials offer advantages but are not yet widely accessible. Proficiency testing data from DLA and FAPAS show that interlaboratory agreement is strongest for unprocessed matrices and decreases in baked, fat-rich, or fermented foods. These findings highlight the need for harmonized validation and for statistical criteria that define acceptable assay performance around the regulatory threshold. Advanced techniques such as liquid chromatography-tandem mass spectrometry provide detailed peptide-level information and can support confirmation in matrices where immunoassays lose sensitivity. A coordinated, performance-based framework aligned with Codex guidance would enable analytical methods to be evaluated against common criteria for recovery, precision, and detection capability. Codex alignment also introduces method dependency, as the current Type I Codex approach for gluten relies on the R5 Méndez ELISA as the defining method. Because Type I methods are empirical defining methods, results are comparable to the definition established by that method rather than necessarily being traceable to an independent reference value for gluten. The use of a single Type I method can therefore limit comparability when laboratories use assays based on other antibodies or extraction and calibration systems, particularly in processed matrices. Integrating such MPCs into regulatory practice, proficiency testing, and private certification systems would improve the comparability of gluten testing results and improve the reliability of gluten-free labeling in international markets.