Specific molecularly imprinted polymers (MIPs) for hypoxanthine (HYP) recognition in aqueous organic media have been developed based upon UV, FTIR and H-1 NMR prepolymerization studies in conjunction with batch rebinding UPLC analyzes. The MIPs, which used the template mimics caffeine (CAF) and theophylline (TPH), are prepared in CHCl3 by one step precipitation polymerization from acrylamide (AM), 2-hydroxyethyl-methacrylate (HEMA) and methacrylic acid (MAA) as functional monomers, whereas ethylene glycol dimethacrylate (EGDMA), divinylbenzene (DVB) and trimethylolpropane triacrylate (TMPTA) as cross-linkers. The magnitude of the pre-polymerization binding constants between TPH and AM, MAA and HEMA is consistent with the complex stoichiometry (1:2 and 1:1) and number of interaction points (3-, 2-, 1-hydrogen bonded motif). The strong (1:2) complex between TPH and AM (K-11 = 3.36 x 10(4) M-1 and K-12 = 1.33 x 10(2) M-1) makes the corresponding MIP the most suitable for HYP recognition. The best performance of the TPH:AM:EGDMA (1:4:20) MIP is reflected in the high IF and high weighted average affinity based on the Freundlich isotherm. Further polymer characterization by ATR-FTIR, elemental analysis, surface area analysis (BET), swelling and SEM yield vital information regarding the degree of polymerization, real monomer:crosslinker ratio, morphology, pore size distribution plus conformational changes on exposure to different solvents. (C) 2015 Elsevier B.V. All rights reserved.