A validated, rapid UPLC–MS/MS method for simultaneous ivabradine, reboxetine, and metoprolol analysis in human plasma and its application to clinical trial samples

► UPLC–MS/MS measurement of ivabradine, reboxetine and metoprolol in human plasma. ► Use of deuterium labeled internal standard for each analyte. ► Validation according to the EMA guideline on bioanalytical method validation. ► Clinical trial samples. ► Measuring patient adherence. A recent clinical trial assessing human autonomic cardiovascular regulation applied pacemaker channel inhibition with ivabradine, norepinephrine transporter blockade with reboxetine, and beta-adrenoreceptor blockade with metoprolol. To verify patient adherence, we developed and validated a fast UPLC–MS/MS assay measuring all three compounds simultaneously. Deuterium-labeled drugs, d3-ivabradine, d5-reboxetine and d7-metoprolol, served as internal standards. Sample preparation of 200μL human plasma consisted of a single liquid–liquid extraction step by means of ethyl acetate. Chromatographic separation was performed on a 50-mm long BEH C18 column with gradient elution using a mixture of water and methanol each containing 2mM ammonium acetate over 4.5min. The mass spectrometer was operated in the positive electrospray ionization (ESI+) mode. Characteristic product ions resulting from collision-induced dissociation of unlabeled and deuterium-labeled drugs with argon were used for quantification in the selected-reaction monitoring mode. We validated the method according to the European Medicines Agency (EMA) guideline on bioanalytical method validation over the range from 1ng/mL to 500ng/mL for all three analytes. Linear responses with correlation coefficients>0.99 over that range were acquired. The LOQ value was 1ng/mL for each drug. Regulatory criteria for accuracy (80–120%) and precision (RSD