3024 J. Sep. Sci. 2014, 37, 3024–3032 Medeea Radulescu 1 Kamil Kuca 2 Kamil Musilek 3 Victor David 1 1 Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest, Bucharest, Romania 2 Biomedical Research Center, University Hospital, Hradec Kralove, Czech Republic 3 Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Czech Republic Received April 7, 2014 Revised July 14, 2014 Accepted August 5, 2014 Research Article Structural modifications of dicationic acetylcholinesterase reactivators studied under ion-pairing mechanism in reversed-phase liquid chromatography A study focused on the chromatographic behavior of several acetylcholinesterase reactivators under ion-pairing mechanism is reported. Among these reactivators, dicationic oximes and carbamoyl-based pyridinium congeners were studied, which form ion pairs with alkylsul- fonate anions. This mechanism was studied for some major experimental parameters, such as the chain length of the ion-pairing agent added to the aqueous phase, its concentration, temperature, and nature of the organic modifier from mobile phase. Retention data showed one or two possibilities of forming ion pairs and the tautomerism of the studied reactivators, for different pH values of the aqueous component. Double sigmoid shapes were obtained for the studied compounds for the dependence between retention factor and pH, indicating the possibility of one or two tautomeric equilibria: at pH close to 7 these compounds are not stable as dicationic species and they participate in the retention process as nitroso forms, which are not able to form ion pairs with alkylsulfonates. The dependences of the retention factor on the organic modifier content from mobile phase were linear. Two complementary theoretical models were used to explain the functional dependences for the retention data on the experimental parameters. Keywords: Adsorption models / Alkylsulfonates / Dicationic oximes / Ion-pairing mechanism / Liquid chromatography DOI 10.1002/jssc.201400390  Additional supporting information may be found in the online version of this article at the publisher’s web-site 1 Introduction Acetylcholinesterase reactivators are compounds successfully used in the therapy of organophosphate intoxication [1–3]. This class comprises mainly oxime-type compounds with pyridinium ring giving the dicationic character [4, 5]. They have a highly hydrophilic character [6], which is considered to limit their distribution through the human body, espe- cially into the central nervous system [7]. A few congeners of this class were determined from various matrices by LC–MS, using diverse retention mechanisms, such as hydrophilic in- teraction, mixed RP/cation exchange [8], and RP ion-pairing (IP) with perfluorinated agents [9, 10]. However, the chro- matographic behavior is tremendously influenced by struc- tural change [11, 12], under chromatographic conditions ap- plied to the elution process. It is the aim of this work to study Correspondence: Professor Victor David, Department of Analyti- cal Chemistry, Faculty of Chemistry, University of Bucharest, Sos. Panduri No.90 Bucharest 050663, Romania E-mail: Vict_David@yahoo.com Abbreviations: HPT, heptane; HXN, hexane; IP, ion-pairing; MeOH, methanol; OCT, octane; PNT, sodium pentane- sulfonate the influence of the main elution parameters on chromato- graphic behavior for several acetylcholinesterase reactivators with dicationic character under IP mechanism due to struc- tural modifications taking place in analyte molecules. This could be useful either for analytical purposes or for a better understanding of the interaction of these compounds with biochemical compounds during their absorption and trans- port as dependent on the structural modification due to the solvation environment [13]. These compounds are interest- ing for studying the IP mechanism due to the fact that they are involved in tautomeric equilibria, which can influence the entire retention process. 2 Materials and methods 2.1 Theoretical framework The ion-pair mechanism is usually applied to very polar compounds (A), which should be present in mobile phase as an ionic species (A ± ) that forms ion pairs with an opposite charged IP agent (IPA ∓ ), added to mobile phase. In our Colour Online: See the article online to view Fig. 2 in colour. C  2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com