High performance liquid chromatographic analysis of the in vitro N-dealkylated and N-oxide metabolites of N-ethyl-N-methylaniline: Methodology for the determination of enzyme activity and stereoselectivity of N-oxidation

摘要

A robust, sensitive reverse-phase high-performance liquid chromatographic (HPLC) method for the quantification of the major in vitro oxidative microsomal metabolites of the prochiral tertiary amine N-ethyl-N-methylaniline (EMA) is described. The analytes were resolved on a Spherisorb 5 ODS1 HPLC column using an acetonitrile/phosphate buffer system under isocratic conditions. Use of solid-phase extraction (SPE) and ultraviolet (UV) detection at low wavelength (210 nm) enabled the detection of EMA N-oxide simultaneously with the major N-dealkylated metabolites, N-ethyl and N-methylaniline. Previous chromatographic methods described for the quantification of N,N-dialkylarylamine N-oxides have generally required sample pretreatment to reduce the N-oxide to its parent tertiary amine prior to analysis. The above method, in combination with a previously reported chromatographic resolution based on the Chiralcel OD chiral stationary phase (CSP), allows accurate and precise determination of both the quantity and stereochemical composition of metabolically derived EMA N-oxide. [References: 20]