Pre-column fluorescence derivatization of peptides containing arginine aldehyde moiety in high-performance liquid chromatography
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A sensitive reversed-phase high-performance liquid chromatographic method with pre-column fluorescence derivatization has been developed for the determination of DLL-MePhe-Pro-Arg-H (LY-DLL, LY-294468). In the search for a derivatization method for the determination of the tripeptide aldehyde, which is present in equilibrium structures in aqueous solution, it was found that the guanidino group of the argininal residue was not converted into a fluorescent derivative by reaction with benzoin. However, if the LY-DLL was first converted into a LY-DLL-TRIS adduct, a fluorescent product could be obtained by the reaction of LY-DLL with TRIS in TRIS(HCl) buffer (pH 8.5) followed by benzoin in the presence of beta-mercaptoethanol and sodium sulphite in an alkaline medium.
Key WordsColumn liquid chromatography Pre-column fluorescence derivatization Arginine-aldehyde peptide LY-294468
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