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

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 C-18 column (150 mm x 4.6 mm, 5 Dikma) using a mixture of methanol and aqueous 5 mM ammonium acetate buffer containing 0.2 A 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 A) 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. (C) 2012 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. Production and hosting by Elsevier B.V. All rights reserved.