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

Formaldehyde is liberated in the process of cytochrome P450 (CYP) mediated demethylation of a wide variety of compounds containing the CH3N or CH3O fu nctionality. A highly sensitive method using a highperformance liquid chrom atography (HPLC) system with postcolumn derivatization was developed to mea sure the liberated formaldehyde as N- and O-demethylase activity of drugs i n human liver microsomes. Following the chromatographic separation of forma ldehyde on a C18 column, the formaldehyde was reacted with the Nash reagent in the postcolumn reactor at 100 degrees C and detected by the fluorescenc e method. The results showed that the present method has excellent precisio n and accuracy. The intra- and interassay variances of this method were les s than 10%. The newly developed HPLC method was found to be about 80-fold m ore sensitive than the colorimetric method in detection of formaldehyde. Th e N-demethylase activity of sertraline in rat liver microsomes determined b y the present method did not differ from those detected by previous methods quantifying produced desmethyl metabolite. The present method has been suc cessfully applied to determine the N-demethylase activities of several drug s, including aminopyrine, erythromycin, fluoxetine, S-mephenytoin, and sert raline, in human liver microsomes. This assay should be useful for generic analysis of N- and O-demethylase activities of xenobiotic and endobiotic ch emicals by CYP enzymes. (C) 2000 Academic Press.