Measurement of methionine enkephalin and leucine enkephalin in rat brain regions by high-performance liquid chromatography with coulometric electrochemical detection

Abstract

A method is described for the determination of two pentapeptides, methionine enkephalin (H-Tyr-Gly-Gly-Phe-Met-OH) (ME) and leucine enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) (LE) in discrete rat brain regions. Separation and quantitation were performed by reversed-phase high-performance liquid chromatography with coulometric detection. Perchloric acid extracts of the tissue after enzyme inactivation by heat treatment were passed through a normal-phase solid-phase extraction diol (COHCOH) column, and endogenous ME and LE were subsequently eluted with methanol. The mobile phase was 1-propanol-phosphate buffer (pH 5.5) (9:91). Eluted samples were detected electrochemically using dual coulometric electrodes operated in screen mode. Each of these enkephalins gave a linear response over the range 40–160 ng/ml cerebellar homogenate (0.8–3.2 ng absolute amount on column). Analytical recoveries of synthetic ME and LE, added to the homogenates, were 70±3 and 70±10%, respectively, when compared with enkephalins dissolved in water. The mean between-assay coefficients of variation for synthetic ME and LE were lower than 10.7 and 7.4%, respectively, over the concentration range studied. The within-assay coefficients of variation for synthetic ME and LE were 11.4 and 9.5%, respectively, at the lowest concentration. The present method has been applied to a study determining the levels of endogenous ME and LE in discrete rat brain regions.