Simultaneous determination of ivabradine, metoprolol and their metabolites in rat plasma by ultra-performance liquid chromatography tandem mass spectrometry and its application in a pharmacokinetic study

Abstract

A sensitive and rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine ivabradine, metoprolol and their corresponding metabolites—N-demethyl ivabradine and α-hydroxy metoprolol in rat plasma simultaneously. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a XEVO TQD triple quadrupole mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 469.2 → 177.2, m/z 455.2 → 262.2, m/z 268.3 → 116.3 and m/z 284.2 → 116.3 were used to quantify ivabradine, N-demethyl ivabradine, metoprolol and α-hydroxy metoprolol, respectively. This assay method has been fully validated in terms of selectivity, linearity, recovery, matrix effects, accuracy, precision and stability. The linearity of this method was found to be within the concentration range of 0.2–100 ng mL⁻¹ for ivabradine, 0.05–20 ng mL⁻¹ for N-demethyl ivabradine, 5.0–5000 ng mL⁻¹ for metoprolol, and 1.0–500 ng mL⁻¹ for α-hydroxy metoprolol in rat plasma, respectively. Only 4.5 min was needed for an analytical run. The method was successfully applied for the analysis of rat plasma samples in their pharmacokinetic study. After oral administration of 1.0 mg kg⁻¹ ivabradine and 10 mg kg⁻¹ metoprolol, the t_1/2 of ivabradine, N-demethyl ivabradine, metoprolol and α-hydroxy metoprolol was (2.12 ± 0.28), (3.41 ± 0.49), (1.93 ± 0.13), and (2.68 ± 0.48) h, respectively.