Accurate quantification of small bioactive peptides derived from dairy and plant-based beverages using isotope-coded dimethyl labeling and LC-MS/MS

Small bioactive peptides found naturally in foods or released during processing or gastrointestinal digestion can exert important biological activity, but their effectiveness depends on concentration, bioaccessibility, and bioavailability. Yet, accurate quantification of these peptides in complex food matrices and digesta remains limited. This study presents a cost-effective method for quantifying small peptides using isotope-coded dimethyl labeling with LC-QqQ MS in multiple reaction monitoring mode. The method was optimized across dairy and non-dairy beverages and validated with soy beverage digesta, selected for its significant protein level. Validation showed high accuracy (91.1–106.0 %), low intra-day precision (≤5.0 %), and inter-day instrumental precision (≤7.3 %) for 24 di- and tripeptides, with LOD and LOQ at sub-ppb levels for most analytes. The method was applied to bovine milk, soy, and almond beverages and their digesta, revealing dynamic peptide levels during digestion. This approach may be extended to quantify peptides in other complex food and biological matrices. • Stable isotope-coded dimethyl labeling enables isotopologue internal standard use. • Validation data confirmed high accuracy and precision for peptide measurement. • Specific bioactive peptides in dairy and plant-based drinks were quantified. • Bioactive peptide levels and trends varied by beverage type and digestion stage.