Measurement of methionine enkephalin and leucine enkephalin in rat brain regions by high-performance liquid chromatography with coulometric electrochemical detection.
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.
Kim
C
C
Addiction Research Foundation, Toronto, Ontario, Canada.
Cheng
R
R
George
S R
SR
D000818:Animals; D001923:Brain Chemistry; D002531:Cerebellum / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D003342:Corpus Striatum / Q000032:analysis; D004563:Electrochemistry; D004743:Enkephalin, Leucine / Q000032:analysis*; D004744:Enkephalin, Methionine / Q000032:analysis*; D007031:Hypothalamus / Q000032:analysis; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D004744:Enkephalin, Methionine; D004743:Enkephalin, Leucine