Bioanalysis of the peptide des-enkephalin-gamma-endorphin. On-line sample pretreatment using membrane dialysis and...

Des-enkephalin-gamma-endorphin is a neuroleptic endogenous peptide that is active in the central nervous system in extremely low concentrations. The pharmacokinetics of this peptide could not be studied in detail as a bioanalytical method for determining endogenous levels of this peptide in biological matrices was not available. Liquid chromatography with fluorescence reaction detection in principle offers sufficient sensitivity for this application, provided that a selective sample pretreatment can be performed. The development of a pretreatment method for plasma samples is described. After protein precipitation with trichloroacetic acid, high-molecular-weight compounds are removed using on-line continuous-flow dialysis. After dialysis, polar low-molecular-weight compounds, including those containing amino functions, are removed by solid-phase isolation, while simultaneously the analyte is concentrated. By means of valve switching the pretreatment system is coupled on-line to the liquid chromatographic system. With the developed system it is possible to determine desenkephalin-gamma-endorphin in plasma in the range 10-100 ng/ml.