Determination of Enantiomeric Purity of Peptides

This technical report outlines a project aimed at developing a reliable method for determining the enantiomeric purity of synthetic peptides using reverse-phase liquid chromatography coupled with accurate mass spectrometry (LC-MS). The report details the significance of enantiomeric purity in peptide therapeutics, emphasizing its impact on efficacy and patient safety. Key challenges such as racemisation during peptide synthesis and analysis are discussed, along with the experimental approach taken to mitigate these issues. The method involved hydrolysing peptides in deuterated hydrochloric acid to accurately quantify D-amino acids and employing Marfey’s reagent for derivatisation, which allows for the separation of enantiomers based on their physical properties. The report presents the optimal conditions identified for hydrolysis and derivatisation, which were found to differ among various peptides. The findings contribute to a deeper understanding of chiral purity testing and establish a foundation for future research in peptide analysis.