Journal of Chromatography A, 1377 (2015) 106–113 Contents lists available at ScienceDirect Journal of Chromatography A jo ur nal ho me pag e: www.elsevier.com/locate/chroma Analytical aspects of achiral and cyclodextrin-mediated capillary electrophoresis of warfarin and its two main derivatives assisted by theoretical modeling Paweł Nowak a , Magdalena Garnysz a , Mariusz Paweł Mitoraj b , Filip Sagan b , Michał Wo´ zniakiewicz a,∗ , Paweł Ko´ scielniak a a Jagiellonian University in Kraków, Faculty of Chemistry, Department of Analytical Chemistry, Kraków, Poland b Jagiellonian University in Kraków, Faculty of Chemistry, Department of Theoretical Chemistry, Kraków, Poland a r t i c l e i n f o Article history: Received 30 July 2014 Received in revised form 7 December 2014 Accepted 9 December 2014 Available online 18 December 2014 Keywords: Acid dissociation constant Capillary electrophoresis pKa shift Cyclodextrins Warfarin Chiral separation a b s t r a c t Several distinct analytical issues have been addressed by performing capillary electrophoresis-based separations of the warfarin, 7-hydroxywarfarin and 10-hydroxywarfarin in an achiral and cyclodextrin- containing media. The measurements were conducted across a range of pH in order to find optimum conditions for achiral and chiral separations. The values of acid dissociation constant (pK a ) have been determined and compared. Subsequently, after performing a series of mobility shift assays at different pH and cyclodextrin concentration, the pK a values ascribed to diastereomeric complexes with methyl- -cyclodextrin have been estimated. The significant pK a shifts upon complexation have been noticed for warfarin – up to 1.5 pH units, and only subtle for 10-hydroxywarfarin. A new approach that allows the estimation of association percentage based on the electrophoretic mobility curves has been also demonstrated. The complex mechanism of chiral separation has been found to be responsible for the observed migration profile, relying on a combined equilibrium between complexation/partition and protonation/deprotonation phenomena. The occurrence of the pK a -related migration order reversal has been demonstrated in achiral medium between warfarin and 7-hydroxywarfarin, and in chiral medium between enantiomers, causing a drop in enantioselectivity at specific pH. In parallel, the density func- tional theory-based calculations have been performed in order to obtain the structures of warfarin and its derivatives as well as to rationalize the shifts in pK a values. © 2014 Elsevier B.V. All rights reserved. 1. Introduction The capillary electrophoresis (CE) is an instrumental sepa- ration technique characterized by high efficiency and general cost-effectiveness, which can be applied for multifarious bioana- lytical purposes. Most commonly CE is used as a separation tool. In particular, it proves to be useful technique for the stereoselective separations [1–9]. Apart from an analysis of samples with constant composition, CE may be used to study the kinetic processes, e.g. to monitor the progress of diverse enzymatic reactions in time, or on the contrary, to measure the dynamics of non-covalent interac- tions in equilibrated mixtures [10–15]. Finally, it also allows one to determine the values of physicochemical parameters, e.g. an acid dissociation constant – pK a , with fairly good accuracy [16]. ∗ Corresponding author at: Jagiellonian University in Kraków, Ingardena 3, 30-060 Kraków, Poland. Tel.: +48 12 663 20 84; fax: +48 12 663 20 84. E-mail address: michal.wozniakiewicz@uj.edu.pl (M. Wo´ zniakiewicz). The efficient separation of enantiomers by CE requires a differ- ence in their effective electrophoretic mobility ( eff ) values, which may result from dynamical interaction of analytes with chiral selec- tor molecules. It has been proven that there are two independent mechanisms which may account for such effect: a different com- plexation percentage caused by unequal affinity of enantiomers, and a different  eff of diastereomeric complexes related to unequal ionization level and/or a different molecular shape [17]. The combined complexation/partition and protonation/deprotonation equilibria correspond to the four distinct subspecies of analyte which may exist, and the final enantioresolution effect is defined by the respective probabilities of analyte to be present in partic- ular form. Therefore, description of chiral separation mechanism based only on the complexation equilibrium may be incomplete, especially when after complexation the enantioselective pK a shifts are observed. The complexation-related selectivity and ionization- related selectivity may cooperate or counteract each other. In some cases, at specific pH or chiral selector concentration val- ues, the enantiomeric migration order reversal (MOR) may occur. http://dx.doi.org/10.1016/j.chroma.2014.12.030 0021-9673/© 2014 Elsevier B.V. All rights reserved.