Quantitation of ten barley-specific amylase/trypsin-inhibitors in the Global EcoSeed panel

Amylase/trypsin-inhibitors (ATIs) are known allergens and triggers of non-celiac wheat sensitivity. Until now, ATIs were only quantitated in wheat species. We developed and validated a targeted stable isotope dilution analysis LC-MS/MS method to quantitate ten barley-specific ATIs, including one monomeric and one dimeric amylase-inhibitor, four chloroform/methanol-soluble types, three subtilisin/chymotrypsin-inhibitors and one amylase/subtilisin-inhibitor. After successful validation in terms of precision, recovery and limits of detection and quantitation, the method was applied to 181 barley accessions from the EcoSeed panel, comprising 113 two-row and 68 six-row barleys of different genetic backgrounds. The overall ATI content was 1.1–5.2 mg/g, corresponding to 0.7–3.6% of the total protein content with no clear distinction between two-row and six-row barleys. This study is the first to provide insights on the ATI content and composition of barley, which can be used to make low-ATI foods for special dietary needs.