Separation and determination of neuropeptides in human plasma by capillary zone electrophoresis

Abstract

The separation efficiencies of seven different neuropeptides, which contain three opioid peptides, were investigated by Capillary Zone Electrophoresis (CZE). The tested neuropeptides were beta-endorphin, methionine-enkephalin, leucine-enkephalin, substance P, somatostatin, vasopressin, and oxytocin. They are related to the pain mechanism in the vertebrate central nervous system. The optimal conditions for separating the neuropeptides were as follows: 30mM citric acid buffer at pH 2.9, 75 mu m (I.D.) X 55 cm fused silica capillary column, and 185 nm wavelength UV detection. The developed CZE method was used to detect the neuropeptides in rat brain tissue homogenate, rat plasma and human plasma. Two neuropeptides, beta-endorphin and substance P, were confirmed in rat brain, and opioid peptides such as beta-endorphin, methionine-enkephalin and leucine-enkephalin were identified in rat plasma. Also, the three opioid peptides were not only identified but also quantitatively analyzed in human plasma. Quantitative analysis was achieved with the use of an internal standard. Recovery after sample preparation was good at 83-91%. Detection limits for these opioid peptides were 0.10 pmol for beta-endorphin and 0.25 pmol for both methionine-enkephalin and leucine-enkephalin.