Simultaneous analysis of verapamil and norverapamil enantiomers in human plasma by high-performance liquid chromatography |
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| Institution: | 1. Centro Nazionale per il Controllo e la Valutazione dei Farmaci, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy;2. Technische Universität Chemnitz, Faculty of Natural Sciences, Institute of Chemistry, Inorganic Chemistry, D-09107, Chemnitz, Germany;3. Technische Universität Chemnitz, Research Center MAIN (Materials, Architecture and Integration of Nanomembranes), Rosenbergstraße 6, D-09126, Chemnitz, Germany;1. NIST Center for Neutron Research, Gaithersburg, MD, United States;2. Rotem Industries Ltd, Rotem Industrial Park, Israel;3. University of Maryland, College park, MD, United States;4. Nuclear Research Center Negev, Beer-Sheva, Israel;5. Eljen Technologies, Sweetwater, TX, United States;1. Department of Pharmacognosy, Semmelweis University, Budapest, H-1085 Üllői út 26, Hungary;2. CycloLab, Cyclodextrin R&D Ltd, Budapest, H-1097 Illatos út 7, Hungary;3. Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43, Prague 2, Czech Republic;1. Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy;2. Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy |
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| Abstract: | An improved HPLC method for the simultaneous determination of the enantiomers of verapamil (V) and its major metabolite norverapamil (NV) in human plasma samples is presented. NV is acetylated immediately to N-acetylnorverapamil (ANV) in the extraction solvent (2% butanol in hexane). Acetylation is so rapid that it does not delay sample processing. ANV and V enantiomers are then separated on an α1-acid glycoprotein chiral column with a mobile phase of phosphate buffer (0.01 M, pH 6.65) and acetonitrile. The fluorescence detector wavelengths are set at 227 nm for excitation and 308 nm for emission. Introduction of the internal standard (I.S.) (+)-glaucine improves accuracy, precision and robustness of the method. The assay is sensitive and specific. Baseline separation is achieved for both V and ANV. Limits of quantitation are 3 ng/ml for V and 2 ng/ml for NV (single enantiomer) with precision and accuracy better than 15% at those levels. Detector response is linear in the range tested (3–200 ng/ml for V and 2–100 ng/ml for NV, single enantiomer). This assay has been applied to a clinical study of the pharmacodynamics of V involving six healthy volunteers. |
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