Radioimmunoassay for the simultaneous determination of morphine and codeine

Antiserum against morphine was produced in rabbits immunized with morphine hapten conjugated to bovine serum albumin. The carrier protein was conjugated to the nitrogen atom of the opiate alkaloid in order to make the phenolic hydroxy group on C3 and the alcoholic group on C6 as determinant groups. The antibody does not recognize codeine or the major metabolite of morphine, 3-O-monoglucuronide. This antibody was used in conjunction with an antibody prepared against 3-O-carboxymethylmorphine to develop a radioimmunoassay which can measure codeine in the presence of morphine. The assay was used to follow both the plasma and brain levels of codeine and its biotransformation to morphine. Codeine when administered at a dose of 5 mg/kg i.v. showed a biphasic plasma decay curve the first phase of which had a +1/2 of 26 min. Peak concentrations of morphine were detected in the plasma following that dose of codeine at 0.5 h. 30 min after the injection of 20 mg/kg i.p. codeine, the brain levels of morphine were only 2% that of codeine. Thereafter, the brain levels of morphine slowly declined.