Determination of Leucine- and Methionine-Enkephalins in Rat Brains by High Performance Liquid Chromatography with Precolumn Fluorescence Derivatization

Fluorometrically reactive peptides of leucine- and methionine-enkephalins in rat brain tissues such as cortex, striatum and hypothalamus were simultaneously assayed by reversed-phase high performance liquid chromatography with fluorometric detection, based on precolumn derivatization specific for N-terminal tyrosine-containing peptides. The enkephalin peptides extracted from the tissues were converted into fluorescent derivatives by reaction with hydroxylamine, cobalt(II) ion and borate in a weakly alkaline aqueous solution (pH 8.5). The fluorescent derivatives of the peptides were separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution of acetonitrile in the mobile phase containing borate buffer (pH 8.5) and tetrabutylammonium chloride, and then determined by fluorometry. The peaks of both the enkephalins in the tissue sample were not observed in the chromatogram after the enzymatic degradation with carboxypeptidase A. The determined concentrations of the leucine- and methionine-enkepalins in the tissues were 20-238 pmol and 80-75 pmol per g of the tissue, respectively. The method was sensitive enough to determine the endogenous enkephalins at concentrations as low as ca. 6 pmol per g of the brain tissues.