Simultaneous quantification of ethylmorphine O-deethylase and N-demethylase activity by high-performance liquid chromatography.

The N-demethylation and O-deethylation of ethylmorphine by cytochrome P-450 are simultaneously measured using high-performance liquid chromatography. All the metabolites and the substrate are extracted from the enzymatic incubation mixture with isopropanol-methylene chloride (20:80) containing 6.0 micrograms/ml codeine sulfate as an internal standard. Separation of the compounds is achieved on a C18 reversed-phase column using a mobile phase of 1% acetic acid--acetonitrile (85:15) with 1-hexanesulfonic acid as a counter-ion. Total run time is 12 min at a flow-rate of 2.0 ml/min and 144 bar. Assay of ethylmorphine N-demethylase and O-deethylase activities in rat liver microsomes revealed close agreement between this method and conventional ones. N-Demethylation was found to greatly exceed O-deethylation in liver microsomes from either control or phenobarbital-treated rats confirming results from other laboratories. This method can also be used to measure the N- and O-demethylation of codeine.