Determination of Seven Opioid Peptides in Rat Brain by High Performance Liquid Chromatography with On-Line Postcolumn Fluorescence Derivatization

Abstract

A high performance liquid chromatographic method with on-line postcolumn fluorescence derivatization is described for a simple and sensitive determination of opioid peptides, including enkephalins in rat brain. The peptides were separated on a reversed-phase column (Asahipak ODP-50) by gradient elution of acetonitrile in the mobile phase containing a borate buffer (pH 10.0). They were then automatically converted into fluorescent derivatives by reactions with hydroxylamine, cobalt(II) ion and borate. This derivatization was specific for N-terminal tyrosine-containing peptides. It was successfully used not only for the simultaneous quantification of the endogenous seven opioid peptides at the picomole level in brain tissue, but also for their identification by means of enzymatic degradation using carboxypeptidase A and trypsin. The detection limits (S/N=3) for the synthetic peptides were 0.5–1.5 pmol in the injection volume.