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

A robust, sensitive reverse-phase high-performance liquid chromatographic ( HPLC) method for the quantification of the major in vitro oxidative microso mal metabolites of the prochiral tertiary amine N-ethyl-N-methylaniline (EM A) is described. The analytes were resolved on a Spherisorb 5 ODS1 HPLC col umn using an acetonitrile/phosphate buffer system under isocratic condition s. Use of solid-phase extraction (SPE) and ultraviolet (UV) detection at lo w wavelength (210 nm) enabled the detection of EMA N-oxide simultaneously w ith the major N-dealkylated metabolites, N-ethyl and N-methylaniline. Previ ous chromatographic methods described for the quantification of N,N-dialkyl arylamine N-oxides have generally required sample pretreatment to reduce th e 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 pre cise determination of both the quantity and stereochemical composition of m etabolically derived EMA N-oxide.