Absolute quantitation of celiac disease-active gluten peptides in gluten-free barley beer by targeted nanoLC-MS/MS

Gluten-free barley beers may cause symptoms in celiac disease (CeD) patients due to CeD-active peptides from partially hydrolyzed gluten. Accurate quantitation of such peptides is essential to improve analytical methods for assessing CeD immunogenic activity and ensuring consumer safety, as routine methods like R5 competitive enzyme-linked immunosorbent assays (R5c ELISA) may miss small yet harmful peptides. This study used targeted nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) with stable isotope dilution assay to quantitate seven CeD-active peptides with complete immunogenic epitopes in 19 gluten-free and four regular or carbohydrate-reduced beers. The selection of CeD-active peptides was made based on their previous identification in the analyzed beers. The beers were subjected to single-pot solid-phase-enhanced sample preparation and chymotryptic hydrolysis. All peptides were under the limit of quantitation in 13 commercially available gluten-free beers. In the remaining six, the total peptide concentrations ranged from 14 to 806 ng/mL, while non-gluten-free beers contained 967–3089 ng/mL. Peptide concentrations correlated with the gluten content measured by R5c ELISA, and lot-to-lot variations were observed. These findings support the need for a more comprehensive risk assessment of products containing partially hydrolyzed gluten.