High-performance liquid chromatography of N-terminal tyrosine-containing oligopeptides by pre-column fluorescence derivatization with hydroxylamine, cobalt(II) and borate reagents

✍ Scribed by Mutsuko Nakano; Masaaki Kai; Masahiro Ohno; Yosuke Ohkura

• Publisher: Elsevier Science
• Year: 1987
• Tongue: English
• Weight: 532 KB
• Volume: 411
• Category: Article
• ISSN: 1873-3778
• DOI: 10.1016/s0021-9673(00)93981-5

✦ Synopsis

A pre-column fluorescence derivatization method is described for the highperformance liquid chromatographic determination of N-terminal tyrosine-containing oligopeptides involving methionine-enkephalin and leucine-enkephalin. The peptides are converted into fluorescent derivatives by heating in a weakly alkaline medium (pH 8.5) containing hydroxylamine, cobalt(I1) ion and borate. The derivatives are separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution of acetonitrile in a mobile phase containing borate buffer (pH 8.5) and tetra-n-butylammonium chloride, and then determined by fluorimetry. The derivatization provides a single fluorescent product for each N-terminal tyrosine-containing oligopeptide, but does not allow the production of fluorescent derivatives for peptides having no tyrosyl residue at the N-terminal. The method is selective and sensitive; the lower limits of detection for the N-terminal tyrosine-containing oligopeptides tested were 14&310 fmol per 100 ~1 injected.