Application of Marfey's reagent in racemization studies of amino acids and peptides

Reversed-phase high-performance liquid chromatography and pre-column derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) were used for monitoring racemization in peptides, amino acids and their derivatives by separation of optical isomers of amino acids. The technique was applied to the analysis of biologically active peptides, amino acids, their N- and C-protected derivatives, branched polypeptides based on polylysine, and endothiopeptides, and to the detection of stereochemical consequences of side reactions and hydrolysis. The chromatographic samples were mixtures of L- and D-amino acids obtained by hydrolysis of different peptides and derivatives. Baseline separations could be achieved on an ODS-Hypersil column with methanol-acetonitrile-acetate buffer (pH 4) mixtures as the eluents. The rates of racemization were calculated.