Simultaneous determination of ivabradine and N-desmethylivabradine in human plasma and urine using a LC-MS/MS method: application to a pharmacokinetic study

Abstract

A sensitive and specific liquid-chromatography tandem mass spectrometry (LC-MS/MS) assay has been developed and validated for the simultaneous quantification of ivabradine and its active metabolite N-desmethylivabradine in human plasma and urine. The assay employed a single liquid–liquid extraction of the analytes from plasma and urine samples, and diazepam was used as internal standard (IS). The chromatographic separation was achieved on a Diamonsil C18 column (150 mm×4.6 mm, 5 μm, Dikma) using a mixture of methanol and aqueous 5 mM ammonium acetate buffer containing 0.2% formic acid (80:20, v/v) as mobile phase. The assay for ivabradine and N-desmethylivabradine in plasma showed good linearity (r≥0.99) over the ranges 0.1013–101.3 ng/mL and 0.085–25.5 ng/mL, respectively. The assay for ivabradine and N-desmethylivabradine in urine showed good linearity (r≥0.99) over the ranges 10.13–6078 ng/mL and 8.5–850 ng/mL, respectively. The intra- and inter-day accuracy and precision values were found to be within the assay variability limits (RSD<15%) in accordance with FDA guidelines. The methods were successfully used for evaluating the pharmacokinetic properties of ivabradine and N-desmethylivabradine in human plasma and urine in Chinese healthy volunteers.

Keywords

• Ivabradine
• N-desmethylivabradine
• Pharmacokinetics
• LC-MS/MS