ESTIMATION OF MORPHINE IN BIOLOGICAL MATERIALS

¹ Department of Pharmacology, University of Michigan Medical School, Ann Arbor
² Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Ind.

Methods of estimation of morphine in biological materials have been described. The procedures involve esterification, presumably of the phenolic hydroxyl of morphine, with p-nitrobenzoyl chloride and extraction of the ester into ethylene dichloride. The p-nitrobenzoyl ester is then estimated in the ethylene dicbloride solution by complexing preferably with methyl orange, or with bromocresol purple. A procedure for the reduction of the nitro group of the ester and subsequent diazotization and coupling with N-(1-naphthyl)ethylenediamine has been established for aqueous solutions of morphine.

The minimum concentration of morphine which can be estimated in plasma is 3 microgm./ml., in urine (concentrated) is 10 microgm./ml, and in tissue is 5 microgm./gm. The precision in plasma is about 95 ± 10 per cent, in urine about 100 ± 5 per cent (the precision is 110 ± 15 per cent with minimum concentrations), and in tissues 100 ± 25 per cent at 5 microgm./gm. and 100 ± 15 per cent at 10 microgm./gm.

Details are also provided for the estimation of "bound" morphine using a standard technique of hydrolysis.

Evidence, based on pH distribution studies and actual recovery of morphine, is presented for the specificity of the method.