Reversed-phase chromatographic behaviour of beta-endorphin: evidence of conformational change.

The effect of alteration in isocratic mobile phase constituents, composition of sample solution, flow-rate and column temperature on the reversed-phase chromatographic behaviour of beta-endorphin was investigated. Beta-Endorphin was shown to be particularly sensitive to the concentration of organic modifier within the mobile phase. The relative contact area of beta-endorphin was demonstrated to be less than that of the much smaller molecule, gamma-endorphin, indicating that beta-endorphin is in a folded form under the mobile phase conditions utilised. Buffer molarity and pH were implicated in the conformational transition of beta-endorphin. In addition, the micro-environment of beta-endorphin prior to its injection onto the high-performance liquid chromatographic (HPLC) column is crucial to its chromatographic behaviour. Manipulation of the sample solvent environment produced reversible conformational modifications ultimately resulting in asymmetric and even split peaks. This phenomenon was more clearly seen when altering HPLC flow-rate. Elevation of HPLC column temperature provided additional evidence of structural change in beta-endorphin, with further conformational forms of this molecule being observed at higher temperatures. This work suggests that the chromatography of beta-endorphin involves a complex mechanism of separation which cannot be adequately explained by the two-state model of kinetic processes.

Measurement of beta-endorphin in human plasma by high-performance liquid chromatography with electrochemical detection: validation of a method employing the simultaneous purification and concentration of beta-endorphin.

The simultaneous purification and concentration of synthetic human beta-endorphin from plasma is described, which when used together with an appropriate isocratic high-performance liquid chromatographic-electrochemical detection (HPLC-ED) system allows the determination of elevated physiological levels of beta-endorphin. Purification of plasma was gained by flash-freezing in liquid nitrogen, acidifying with 100 microliters of trifluoroacetic acid (10%, v/v) per ml of plasma, thawing at 4 degrees C and centrifuging to remove any precipitate. Solid-phase extraction with silica sorbent was utilised, which allowed further isolation of the analyte, a method of concentration and a procedure whereby beta-endorphin could be transferred to the HPLC mobile phase. Silica sorbent demonstrated greater selectivity than C18 for synthetic human beta-endorphin and, in addition, provided improved recovery of this analyte when utilising elution volumes of 500 microliters or less. Proteolytic degradation and heparin-induced high-affinity binding in plasma were shown not to effect the recovery of beta-endorphin if blood was rapidly chilled and plasma quickly obtained, frozen and acidified. Validation of this purification/concentration method using [125I]beta-endorphin demonstrated a recovery of 85.6% which was not jeopardised when concentrating the sample twenty-fold. This provided an increase in the sensitivity of detection, when used in conjunction with HPLC-ED, from 5 ng/ml to 250 pg/ml.