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A strict gluten-free diet is the only effective therapy for celiac disease (CD) patients. Currently, enzyme-linked immunosorbent assays (ELISA) are most commonly used for gluten analysis to monitor the safety of gluten-free products, but these assays primarily target the alcohol-soluble prolamin fraction of gluten. The gluten content is then calculated by multiplying the prolamin content by a factor of 2. The problem is that different types of grains contain variable proportions of prolamins and alcohol-insoluble glutelins. As a result the calculated gluten content may be either over- or underestimated, which is a food safety issue for CD patients. The aim of the present study was to develop a new independent non-immunochemical method for the quantitation of prolamins and glutelins (= total gluten) by liquid chromatography – tandem mass spectrometry (LC-MS/MS). First, protein fractions (prolamins and glutelins) and protein types from wheat, rye, barley and oat flour mixtures were isolated and purified to serve as defined reference proteins. All isolated proteins (fractions and types) as well as the flours were partially hydrolyzed with chymotrypsin and analyzed by LC-MS/MS. Several peptides were identified which are specific indicators for each protein type (marker peptides) and suitable for gluten quantitation. Two to three marker peptides were selected for each protein type and will be quantitated by a stable isotope dilution assay. Then, peptide concentrations can be converted to protein concentrations, because the yield of peptides obtained from a given amount of protein is known. The sum of all protein concentrations will result in the true gluten content of the food sample. This versatile new method will allow the accurate detection of gluten especially in processed or hydrolyzed products where many ELISA have been shown to underestimate gluten contents.