Novel sensitive high-performance liquid chromatographic method for assay of coumarin 7-hydroxylation
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Updated: Oct 18, 2026

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example
Published on: November 18, 2015
1Biotransformations Group, Center of Occupational Diseases, National Institute of Public Health, Prague, Czech Republic. pavel.soucek@ecn.cz
Abstract:
In this paper, a novel HPLC-based method with fluorometric detection of coumarin 7-hydroxylase is presented. The described method provides a time-effective, more sensitive and specific alternative to the previously used spectrofluorometric assay. Using the developed method, metabolism of coumarin in 11 samples of human liver microsomes was evaluated and 1790+/-690 pmol/min/nmol cytochrome P450 (CYP) activity was found. Kinetic parameters and linearity of coumarin 7-hydroxylation were studied in a reconstituted system consisting of recombinant CYP2A6 expressed in Escherichia coli, rat NADPH-CYP reductase and usual components. It was found that a 3.5 to 30 min time of incubation is suitable for estimation of coumarin 7-hydroxylase activity. Observed Km and Vmax values in the CYP2A6 reconstituted system were 1.48+/-0.37 microM and 3360+/-180 pmol product/min/nmol CYP, respectively.
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