Investigation of ex vivo stability of fesoterodine in human plasma and its simultaneous determination together with its active metabolite 5-HMT by LC–ESI-MS/MS: Application to a bioequivalence study

Fesoterodine is a non-selective muscarinic-receptor antagonist, used in the treatment of overactive bladder syndrome. A highly sensitive, selective and rapid method has been developed for the simultaneous determination of fesoterodine and its active metabolite, 5-hydroxymethyl tolterodine (5-HMT) in human plasma by liquid chromatography–tandem mass spectrometry (LC–ESI-MS/MS). Due to rapid conversion of parent drug to 5-HMT, ex vivo stability of fesoterodine in human plasma was extensively studied to optimize the extraction protocol. The analytes and their deuterated analogs were quantitatively extracted from 100 μL human plasma by liquid–liquid extraction in methyl tert-butyl ether: n-hexane. The chromatographic separation of analytes was achieved on a Kromasil C18 (100 mm × 4.6 mm, 5 μm) column under isocratic conditions. The method was validated over a dynamic concentration range of 0.01–10 ng/mL for both the analytes. Ion-suppression effects were investigated by post-column infusion of analytes. The precision (% CV) values for the calculated slopes of calibration curves, which would reflect the relative matrix effect, were less than 1.5% for both the analytes. The intra-batch and inter-batch precision (% CV) across quality control levels varied from 1.82 to 3.73% and the mean extraction recovery was >96% for both the analytes. The method was successfully applied to a bioequivalence study of 8 mg fesoterodine tablet formulation (test and reference) in 12 healthy Indian subjects under fasted and fed condition. The assay reproducibility estimated by reanalysis of incurred samples showed a change of ±12.0%.

► First report on simultaneous determination of FESO and 5-HMT in human plasma.
► Thorough investigation of ex vivo stability of fesoterodine in plasma samples.
► Mitigation of FESO degradation by pre-treatment of blood with sodium metabisulphite.
► The method is practically free of endogenous/exogenous matrix interference.
► Bioequivalence study in healthy Indian volunteers and incurred sample reanalysis.