A fluorometric method for measuring ethoxycoumarin O-deethylase activity by reversed-phase high performance liquid chromatography

Abstract

A high-performance liquid chromatographic (HPLC) method that uses an on-line change in the protonation state of the nonfluorescent product to yield a fluorescent derivative that is detected by fluorometry was developed for the determination of 7-ethoxycoumarin O-deethylase activity. Tissue samples (1-20 micrograms protein) were incubated with 7-ethoxycoumarin, and 7-hydroxycoumarin metabolite was extracted in chloroform. Following drying under nitrogen, the extract was resuspended in methanol (10-100 microliters) and an aliquot of 5-20 microliters was directly injected into a C8 Nova-Pak column. Isocratic separation of hydroxycoumarin was achieved using a mobile phase consisting of methanol:1% acetic acid, 35:65, v/v, pH 3.5, at a flow rate of 1 ml/min. Following chromatographic separation, samples were derivatized with 1.0 N NaOH prior to fluorescent measurements. The detection limit for 7-hydroxycoumarin was less than 1 pmol, with a mean recovery from the incubates of 96.4 +/- 2.3%. This HPLC-fluorometric method was linear up to at least 400 pmol of 7-hydroxycoumarin and could accurately detect metabolite formation in incubates containing control liver microsomes with less than 0.05 microgram total protein. The method also allowed determinations of cytochrome P450-dependent function in extrahepatic tissues of rats, including individual segments of gastrointestinal epithelium and brain, as well as in cultured cells, such as HepG2 cells, in which microsomal protein yield is very small. The wide range of linearity afforded by this method allows a reliable estimation of cytochrome P450-dependent function in samples containing varying concentrations of protein.