Efforts to localize and quantify endorphins require techniques that are highly sensitive and specific. Current assays including bioassays, radioreceptor, radioimmunoassay (RIA) and high performance liquid chromatography (LC) with UV detection lack either adequate specificity or adequate sensitivity, particularly for the study of the larger endorphins. LC has been used to separate endorphins in biological samples but quantification is performed by RIA for every endorphin in several chromatographic fractions. The purpose of this project is to develop a specific and quantitative method for the assay of endorphins from small brain regions using LC with electrochemical detection (LC-EC). To accomplish this, the sensitivity of EC detection will be improved using a variety of procedures including alternative electrode configurations (conventional glass carbon, graphite composite, and reticulated vitreous carbon), variation in electrode surface preparation, electrode pre-treatment with anodization procedures and lastly chemical derivatization procedures to improve endorphin electroactivity. The LC-EC assay for endorphins will then be evaluated by several analytical techniques including hydrodynamic voltammetry, peak current ratios, RIA and mass spectrometry to corroborate peak purity and peak identity. The effect of amino acid sequence upon the electroactivity of the N-terminal tyrosyl will be evaluated in a variety of dipeptides and longer peptide sequences to examine both steric and inductive effects upon redox activity. Met- and leu-enkephalin, the dynorphins and -endorphins will be extracted from specific brain regions and the quantitative values obtained with the LC-EC assay compared with those obtained by RIA of the samples. This LC-EC assays will be especially useful for those studies which require a detailed analysis of endorphin content.
