title: High-performance liquid chromatography determination of N- and O-demethylase activities of chemicals in human liver microsomes: application of postcolumn fluorescence derivatization using Nash reagent.
abstract: Formaldehyde is liberated in the process of cytochrome P450 (CYP) mediated demethylation of a wide variety of compounds containing the CH(3)N or CH(3)O functionality. A highly sensitive method using a high-performance liquid chromatography (HPLC) system with postcolumn derivatization was developed to measure the liberated formaldehyde as N- and O-demethylase activity of drugs in human liver microsomes. Following the chromatographic separation of formaldehyde on a C18 column, the formaldehyde was reacted with the Nash reagent in the postcolumn reactor at 100 degrees C and detected by the fluorescence method. The results showed that the present method has excellent precision and accuracy. The intra- and interassay variances of this method were less than 10%. The newly developed HPLC method was found to be about 80-fold more sensitive than the colorimetric method in detection of formaldehyde. The N-demethylase activity of sertraline in rat liver microsomes determined by the present method did not differ from those detected by previous methods quantifying produced desmethyl metabolite. The present method has been successfully applied to determine the N-demethylase activities of several drugs, including aminopyrine, erythromycin, fluoxetine, S-mephenytoin, and sertraline, in human liver microsomes. This assay should be useful for generic analysis of N- and O-demethylase activities of xenobiotic and endobiotic chemicals by CYP enzymes.
authors.affiliation: Department of Biochemical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Chiba, 263-8522, Japan. kaoruk@p.chiba-u.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Kobayashi
authors.forename: T
authors.initials: T
authors.lastname: Yamamoto
authors.forename: M
authors.initials: M
authors.lastname: Taguchi
authors.forename: K
authors.initials: K
authors.lastname: Chiba
mesh_terms: D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D010090:Oxidoreductases, O-Demethylating / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015262:Xenobiotics / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D015262:Xenobiotics; D010090:Oxidoreductases, O-Demethylating; D010089:Oxidoreductases, N-Demethylating