Bioanalysis of the neuropeptide des-enkephalin-gamma-endorphin by high-performance liquid chromatography with on-line sample pretreatment using gel permeation and solid-phase isolation.
A bioanalytical method is described that allows the determination of a number of beta-endorphin-related peptides. The method is based on the application of fluorescence detection after high-performance liquid chromatography followed by post-column derivatization with o-phthaldialdehyde. Concentrations exceeding 10-25 ng/ml could be determined by using conventional fluorescence detection, whereas lower concentrations demand the use of laser-induced fluorescence detection. The sample pretreatment includes the use of on-line gel permeation, on-line solid-phase isolation and heart cutting of a peak from reversed-phase gradient elution. The sample pretreatment procedure does not discriminate between the dodecapeptide des-enkaphalin-gamma-endorphin (DE gamma E) and its metabolites in order to obtain similar recoveries for all components. The final chromatographic phase system is based on ion-pair formation, which permits the separation of DE gamma E from its metabolites and degradation products. The optimized procedure allows the determination of these peptides in plasma at concentration levels down to about 1 ng/ml, demanding a sample volume of 1 ml.
Stegehuis
D S
DS
Division of Analytical Chemistry, University of Leiden, Netherlands.
Tjaden
U R
UR
van den Beld
C M
CM
van der Greef
J
J
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D004355:Drug Stability; D006801:Humans; D001615:beta-Endorphin / Q000097:blood*
D001615:beta-Endorphin; C033558:des-enkephalin-gamma-endorphin