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

Abstract

The simultaneous purification and concentration of synthetic human β-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 β-endorphin. Purification of plasma was gained by flash-freezing in liquid nitrogen, acidifying with 100 μl of trifluoroacetic acid (10%, v/v) per ml of plasma, thawing at 4°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 β-endorphin could be transferred to the HPLC mobile phase. Silica sorbent demonstrated greater selectivity than C 18 for synthetic human β-endorphin and, in addition, provided improved recovery of this analyte when utilising elution volumes of 500 μl or less. Proteolytic degradation and heparin-induced high-affinity binding in plasma were shown not to effect the recovery of β-endorphin if blood was rapidly chilled and plasma quickly obtained, frozen and acidified. Validation of this purification/concentration method using [ 125I]β-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.