Differentiation between isomeric oxidative metabolites of a piperidine-containing drug by liquid chromatography-tandem mass spectrometry with stable-isotope tracer technique.

This report describes the application of a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to differentiation of hydroxylations and N-oxidations and of two different aliphatic hydroxylations in the investigation of the metabolism of pibutidine hydrochloride, a novel H2 antagonist, the structure of which includes a piperidine ring. Pibutidine metabolites in urine samples from adult male volunteers after oral administration of pibutidine hydrochloride were separated by reversed-phase LC and ionized using an electrospray ionization (ESI) interface. A hydroxylated form of pibutidine was distinguished from the N-oxide by comparison of their product ion spectra, although their mass-to-charge ratios of protonated molecules were identical. Further, two hydroxylated compounds were present in rat microsomal incubation mixtures with pibutidine. The distinction between their positions of hydroxylation (β- and γ-carbon hydroxylation) on the piperidine ring was studied using [piperidine-2H10] pibutidine as incubation substrate. The production of the β-hydroxylated form was accompanied by the elimination of three 2H, resulting from a mechanism including the formation of iminium/enamine. The participation of the iminium ion intermediate in the β-hydroxylation was confirmed by the observation that a cyanide adduct of pibutidine was formed instead of the β-hydroxylated form when another incubation was performed in the presence of cyanide. Copyright © 2001 John Wiley & Sons, Ltd.