Gluten analysis faces challenges in processed foods, requiring harmonized methods for 20 mg/kg regulatory threshold.

For individuals with celiac disease (CD), an autoimmune enteropathy triggered by proline-rich gluten immunogenic peptides (GIPs), a strict gluten-free diet is the only effective treatment. Poorly controlled CD can lead to severe complications like osteoporosis, malnutrition, and small bowel lymphoma. To ensure consumer safety and enable effective dietary management, robust analytical systems are crucial for reliably measuring gluten in diverse food products. Current standard-of-care relies on accurate food labeling, but the molecular complexity of gluten and processing-induced changes pose significant analytical hurdles.

This comprehensive review examined the scientific, analytical, and regulatory factors influencing gluten quantification to support robust enforcement of the 20 mg gluten/kg threshold. It focused on method performance criteria (MPCs) for various analytical systems. The review analyzed how processing steps like baking, fermentation, and extrusion alter gluten extractability and antibody recognition. It also evaluated the performance of different enzyme-linked immunosorbent assays (ELISAs) using various antibodies, extraction chemistries, and calibration standards, including PWG gliadin and incurred reference materials. The analysis incorporated proficiency testing data from DLA and FAPAS to assess interlaboratory agreement across different food matrices.

The review identified significant challenges in accurately quantifying gluten, particularly in processed foods. The molecular complexity of gluten and structural changes from baking, fermentation, and other processing steps profoundly influence extractability and epitope availability. This leads to variable results across ELISAs that employ different antibodies and extraction methods. While the widespread use of PWG gliadin as a calibrant supports harmonized calibration for wheat-based analyses, it can introduce bias as it doesn't reflect 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 revealed that interlaboratory agreement is strongest for unprocessed matrices but significantly decreases in baked, fat-rich, or fermented foods.

These findings highlight a critical need for harmonized validation and statistical criteria to define acceptable assay performance around the regulatory 20 mg gluten/kg threshold. Advanced techniques like liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide detailed peptide-level information, offering potential for confirmation in matrices where immunoassays lose sensitivity.