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.
E J
EJ
Jarvi
J C
JC
Stolzenbach
R E
RE
Larson
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005037:Ethylmorphine-N-Demethylase / Q000032:analysis*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000032:analysis*; D010089:Oxidoreductases, N-Demethylating / Q000032:analysis*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C020130:ethylmorphine O-deethylase; D010089:Oxidoreductases, N-Demethylating; D005037:Ethylmorphine-N-Demethylase; D010634:Phenobarbital