Improved high-performance liquid chromatographic method for the determination of ethylmorphine and its metabolites in microsomal incubations and cell culture media.
Ethylmorphine N-demethylation is used as a marker pathway in studies of rat cytochrome P450 3A and 2C11 biotransformations. At present, microsomal activities are generally measured by a colorimetric determination of the formed formaldehyde. In the present study, a high-performance liquid chromatographic method of separating and quantifying both the N-demethylated (norethylmorphine) and the O-de-ethylated (morphine) metabolites is described. Either samples are extracted with ethyl acetate or proteins are precipitated with zinc sulphate-barium hydroxide. Separation is achieved on a CN reversed-phase column, using a mobile phase of phosphate buffer (pH 4.5)-acetonitrile (90:10, v/v). At a flow-rate of 1.5 ml/min, the analysis time is 30 min. The limit of detection (ultraviolet, 210 nm) for ethylmorphine and its metabolites is 0.5 micrograms/ml.
van't Klooster
G A
GA
Department of Veterinary Basic Sciences, University of Utrecht, Netherlands.
Woutersen-van Nijnanten
F M
FM
Kolker
H J
HJ
Noordhoek
J
J
van Miert
A S
AS
D000085:Acetates; D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003470:Culture Media / Q000737:chemistry*; D005036:Ethylmorphine / Q000032:analysis* / Q000378:metabolism*; D006041:Goats; D008099:Liver / Q000737:chemistry / Q000166:cytology*; D008862:Microsomes, Liver / Q000737:chemistry*; D009020:Morphine / Q000032:analysis
D000085:Acetates; D003470:Culture Media; C007650:ethyl acetate; D009020:Morphine; D005036:Ethylmorphine