Retention and molecular-recognition mechanisms of molecularly imprinted polymers for warfarin derivatives and their application for the determination of warfarin in human serum

Abstract

Monodisperse molecularly imprinted polymers (MIPs) for warfarin (WF), 4ʹ-chlorowarfarin (CWF), 4ʹ-bromowarfarin (BWF), 4ʹ-nitrowarfarin (NWF) and 4ʹ-methylwarfarin (MWF) (MIPWF, MIPCWF, MIPBWF, MIPNWF and MIPMWF, respectively) were prepared using 4-vinylpyridine (4-VPY) and ethylene glycol dimethacrylate as a functional monomer and crosslinker, respectively, by multi-step swelling and polymerization. The retention and molecular-recognition properties of those MIPs were evaluated in HILIC, and reversed- and normal-phase modes. According to 1H NMR studies, one-to-three complex formation of one WF or CWF molecule with three 4-VPY molecules occurred. Via computational approaches, the intermolecular interaction modes and energies between WF derivatives and 4-VPYs were evaluated by semi-empirical quantum chemistry methods and density functional theory calculations. Three major possible hydrogen bonding interaction modes were identified: the interactions between the 4-hydroxy group, α-proton (methylene C–H) and α-proton (methyl C–H) of the WF derivative and the nitrogen atoms of 4-VPYs. In HILIC and normal-phase modes, the interaction energies showed satisfactory correlations with the retention factors of the WF derivatives. In reversed-phase mode, the retention factors of the WF derivatives were described by the hydrophobicity and the acidity of the 4-hydroxy groups of the WF derivatives. These results demonstrate that three hydrogen bonding interactions in HILIC and normal-phase modes, and hydrogen bonding or ionic interactions and hydrophobic interactions in reversed-phase mode play important roles in the retention and molecular-recognition of the WF derivatives on MIPs. Furthermore, MIPBWF was successfully applied to the determination of WF in human serum by column-switching LC with high accuracy, precision and selectivity and without template-leakage problems.