A rapid and sensitive quantitation method of endogenous morphine in human plasma

Endogenous morphine is found at very low levels (pg/ml) in human plasma. Without technical modification, the usual techniques, i.e., RIA or HPLC, can not directly detect its presence. As a result the initial purification process is very crucial. In this report we demonstrate this opiate alkaloids determination in 2 ml of human plasma. Following an acidified precipitation of proteins in plasma, a solid-phase extraction, Sep-pak collection, was employed to concentrate the endogenous morphine. The extract was dissolved in a pH 8.9 phosphate buffer and then extracted into an organic solvent (chloroform and isopropyl alcohol). The initially purified sample was separated by HPLC and detected with an electrochemical detector of 500 mV and 0.02 Hz. The results showed that the recovery of morphine by this technique was 86 + 5.7%, and the sensitivity was 50 pg/ml. Compared to the detection sensitivity of the HPLC (2 ng/ml), this method increased the detection limit by 40-fold. With this method the concentration of endogenous morphine in plasma was determined as 80 pg/ml.