title: A validated, rapid UPLC-MS/MS method for simultaneous ivabradine, reboxetine, and metoprolol analysis in human plasma and its application to clinical trial samples.
abstract: 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<15%) were met for all drugs. The internal standard-normalized matrix factor was close to 1 for low and high analyte concentrations. We successfully measured ivabradine, reboxetine, and metoprolol concentrations in 107 human plasma samples from a clinical trial. Quality control samples processed in parallel confirmed the method's reliability in a clinical setting.
authors: Institute of Clinical Pharmacology, Hannover Medical School, Hannover, Germany. zoerner.alexander@mh-hannover.de
authors: Alexander A
authors: AA
authors: Zoerner
authors: Christoph
authors: C
authors: Schroeder
authors: Arslan Arinc
authors: AA
authors: Kayacelebi
authors: Maria T
authors: MT
authors: Suchy
authors: Frank-Mathias
authors: FM
authors: Gutzki
authors: Dirk O
authors: DO
authors: Stichtenoth
authors: Jens
authors: J
authors: Tank
authors: Jens
authors: J
authors: Jordan
authors: Dimitrios
authors: D
authors: Tsikas
mesh_terms: D001552:Benzazepines / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018592:Cross-Over Studies; D004311:Double-Blind Method; D004355:Drug Stability; D006801:Humans; D000077550:Ivabradine; D016014:Linear Models; D008297:Male; D008790:Metoprolol / Q000097:blood*; D009025:Morpholines / Q000097:blood*; D016032:Randomized Controlled Trials as Topic; D000077593:Reboxetine; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001552:Benzazepines; D009025:Morpholines; D000077550:Ivabradine; D000077593:Reboxetine; D008790:Metoprolol