GRADIENT HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ANALYSIS OF ENKEPHALIN PEPTIDES, THEIR METABOLITES AND ENZYME-INHIBITORS USING COMBINED ULTRAVIOLET AND ELECTROCHEMICAL DETECTION .1. OPTIMIZATION OF SEPARATIONAND DETECTION

Coulometric electrochemical detection (ED) was compatible with gradien t HPLC analysis of enkephalin peptides, their metabolites and several enzyme inhibitors with combined UV detection. The compounds were separ ated using a reversed-phase octylsilane column with 35 mM phosphate bu ffer (pH 2.1)-acetonitrile eluent. Hydrodynamic voltammograms were det ermined for [D-Ala(2)]-methionine enkephalinamide (MEA) and its metabo lites, and the response of MEA consisted of the oxidation of the tyros ine and methioninamide residues with a total transfer of five electron s at the highest potential used. Based on the voltammograms, ED was op timized for the selective determination of MEA and other tyrosine-cont aining peptides with detection limits between 0.4 and 0.6 pmol per 50 mu l injection. In addition, simultaneous UV detection at 205 nm allow ed the determination of all the aromatic peptides and enzyme inhibitor s with detection limits between 1.5 and 30.0 pmol.