Development and validation of a sensitive LC-MS/MS-ESI method for the determination of ivabradine in human plasma: application to a pharmacokinetic study.

A sensitive, rapid assay method for estimating ivabradine in human plasma has been developed and validated using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The procedure involved extraction of ivabradine and the internal standard (IS) from human plasma by solid-phase extraction. Chromatographic separation was achieved using an isocratic mobile phase (0.1% formic acid-methanol, 60:40, v/v) at a flow rate of 1.0 mL/min on an Aglient Eclipse XDB C8 column (150 × 4.6 mm, 5 µm; maintained at 35 °C) with a total run time of 4.5 min. Detection was achieved using an Applied Biosystems MDS Sciex (Concord, Ontario, Canada) API 3200 triple-quadrupole mass spectrometer. The MS/MS ion transitions monitored were 469-177 for ivabradine and 453-177 for IS. Method validation was performed according to Food and Drug Administration guidelines, and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 0.1-200 ng/mL. The lower limit of quantitation achieved was 0.1 ng/mL. Intra- and inter-day precisions were in the range of 1.23-14.17% and 5.26-8.96%, respectively. Finally, the method was successfully used in a pharmacokinetic study that measured ivabradine levels in healthy volunteers after a single 5 mg oral dose of ivabradine.