Complementary and efficient methods for di- and tri-peptide analysis and amino acid quantification from simulated gastrointestinal digestion of collagen hydrolysate

Abstract Collagen hydrolysates (CHs) are composed of bioactive peptides (BAPs) and amino acids (AAs), which contribute to their health enhancing properties. Post digestion profiling of CHs typically evaluates either BAP or AA content in blood but not within digests. Existing methods for peptide analysis are optimized for blood samples and rely on costly methods that require substantial sample preparation and data interpretation. A capillary electrophoresis (CE) method was developed as a rapid, cost effective, and reliable method for analysis of BAPs (Ala-Hyp, Pro-Hyp, Pro-Hyp-Gly, Gly-Pro-Hyp) within digests. Coupled to LC-MS, a hydrophilic interaction liquid chromatography (HILIC) column was used to quantify 19 AAs in digests, without derivatization. Two bovine CHs (CH-GL and CH-OPT) underwent in vitro digestion and their BAP and AA content was assessed. The Gly-Pro-Hyp and Pro-Hyp-Gly content was greater in CH-GL versus CH-OPT with values of 19.82 ± 4.25 and 8.969 ± 2.742 μg/mL respectively. The two CHs had distinct peptide profiles; 13 unidentified peptide peaks from each CH were not found in the other. No differences in AA content were observed. The present work describes sensitive and rapid methodology for concurrent analysis of BAPs and AAs after digestion of CHs, which can support further understanding of the bioactive components of CHs.