Determination of Ivabradine and its Main Metabolite in Rat Plasma by UPLC-MS/MS and its Application to Pharmacokinetics

A sensitive and rapid high-performance liquid chromatography-mass spectrometry method (UPLC-MS/MS) was developed to determine ivabradine and its main metabolite: N-demethyl ivabradine in rat plasma using carbamazepine as the internal standard (IS). The analytes and IS were separated on a Waters ACQUITY BEH C18 column (50 mm x 2.1 mm i.d., 1.7 mu m particle size) with a total run time of 3.0 min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at 469.3 -> 177.2 for ivabradine, 455.2 -> 262.2 for N-demethyl ivabradine and 237.1 -> 194.2 for IS. The calibration curves were linear over the 0.2-100 ng/mL for ivabradine and 0.05-25 ng/mL for N-demethyl ivabradine in plasma, respectively. Relative standard deviation (RSD) of intra-day and inter-day precision were in the range of 2.36%-12.39% for ivabradine and 6.25%-13.51% for N-demethyl ivabradine, respectively. The developed method was successfully applied to pharmacokinetic studies after single intragastric administration of ivabradine 0.8 mg/kg to rats.