High-performance liquid chromatography of N-terminal tyrosine-containing oligopeptides by pre-column fluorescence derivatization with hydroxylamine, cobalt (II) and borate reagents.
A pre-column fluorescence derivatization method is described for the high-performance 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(II) 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 140-310 fmol per 100 microliters injected.
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
M
M
Nakano
M
M
Kai
M
M
Ohno
Y
Y
Ohkura
D001888:Boric Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000032:analysis; D006898:Hydroxylamines / Q000032:analysis; D007202:Indicators and Reagents; D009842:Oligopeptides / Q000032:analysis*; D011863:Radioimmunoassay; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000032:analysis*
D001888:Boric Acids; D006898:Hydroxylamines; D007202:Indicators and Reagents; D009842:Oligopeptides; D003035:Cobalt; D014443:Tyrosine