Automated high-performance liquid chromatographic assay for the determination of 7-ethoxycoumarin and umbelliferone.
An improved high-performance liquid chromatographic assay is presented for the determination of 7-ethoxycoumarin O-deethylase activity. Following a 30-min microsomal incubation, 7-ethoxycoumarin, 4-methylumbelliferone (internal standard), and the metabolite umbelliferone were extracted with chloroform. Separation was achieved with an isocratic mobile phase using a microBondapak phenyl (300 mm x 3.9 mm I.D.) analytical column. The effluent was monitored by fluorescence detection with an excitation wavelength of 360 nm and an emission wavelength of 470 nm. The intra- and inter-assay coefficients of variation were 10 and 6%, respectively. A detection limit of 0.07 micrograms/ml was achieved, making this method suitable for characterizing P-450 activity of human livers.
Evans
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Pharmaceutical Division, St. Jude Children's Research Hospital, Memphis, TN 38101.
Relling
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D015660:7-Alkoxycoumarin O-Dealkylase / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003374:Coumarins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D014468:Umbelliferones / Q000032:analysis* / Q000378:metabolism
D003374:Coumarins; D014468:Umbelliferones; C017299:7-ethoxycoumarin; C031477:7-hydroxycoumarin; D015660:7-Alkoxycoumarin O-Dealkylase