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.
Monger
L S
LS
Pharmaceutical Sciences Research Group, Brighton Polytechnic, Moulsecoomb, E. Sussex, UK.
Olliff
C J
CJ
D007611:Aprotinin; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D004743:Enkephalin, Leucine / Q000097:blood / Q000302:isolation & purification; D004744:Enkephalin, Methionine / Q000097:blood / Q000302:isolation & purification; D006493:Heparin; D006801:Humans; D015203:Reproducibility of Results; D013696:Temperature; D001615:beta-Endorphin / Q000097:blood* / Q000302:isolation & purification
D004744:Enkephalin, Methionine; D004743:Enkephalin, Leucine; D001615:beta-Endorphin; D006493:Heparin; D007611:Aprotinin