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A liquid chromatographic column containing immobilized alpha3beta4 nicotinic acetylcholine receptors (alpha3beta4-nAChRs) has been used to determine the equilibrium association constants (Ka), desorption rate constants (kd), and adsorption rate constants (ka) for the noncompetitive inhibitors: mecamylamine, ketamine, bupropion, and dextromethorphan. Displacement chromatography, with mecamylamine as the displacer, was used to verify that the four compounds bound to the same site on the immobilized alpha3beta4-nAChRs. Nonlinear chromatographic techniques were then utilized to calculate the Ka, ka, and kd values associated with the formation of the noncompetitive inhibitor-alpha3beta4-nAChR complexes. The ka values determined in this study ranged from 19.7 to 10.5 microM(-1) sec(-1), with a relative order of mecamylamine > dextromethorphan > or = ketamine > bupropion. The kd values determined in this study indicated that dextromethorphan-induced inhibition should produce a longer recovery time than the other three NCIs. This was consistent with results from a previous in vitro study. The data from this study indicate that the immobilized alpha3beta4-nAChR column and nonlinear chromatography can be used in the study of NCIs at the alpha3beta4-nAChR.

Uniformly sized molecularly imprinted polymers (MIPs) for (S)-nilvadipine have been prepared by a multistep swelling and polymerization method using methacrylic acid, 2-(trifluoromethyl)acrylic acid, 2-vinylpyridine, or 4-vinylpyridine (4-VPY) as a functional monomer and ethylene glycol dimethacrylate (EDMA) as a cross-linker. The chiral recognition abilities of the MIPs for nilvadipine and other dihydropyridine calcium antagonists were evaluated using a mixture of sodium phosphate buffer (or water) and acetonitrile or only acetonitrile as the mobile phase. The (S)-nilvadipine-imprinted 4-VPY-co-EDMA polymers gave the highest resolution for nilvadipine among the MIPs prepared. In addition, the enantioseparation of nilvadipine was attained using the (S)-nilvadipine-imprinted EDMA polymers, without use of a functional monomer. 1H NMR and molecular modeling studies suggested a one-to-one hydrogen-bonding-based complex formation of (S)-nilvadipine with 4-VPY in chloroform. These results reveal that the (S)-nilvadipine-imprinted EDMA polymers could recognize the template molecule by its molecular shape, and that in addition to this recognition, hydrophobic and hydrogen-bonding interactions seems to play important roles in the retention and chiral recognition of nilvadipine on the 4-VPY-co-EDMA polymers in hydroorganic mobile phases. By optimizing chromatographic conditions such as column temperature and flow rate, the baseline separation of nilvadipine enantiomers was attained with a short analysis time and with a column efficiency comparable to commercially available chiral stationary phases based on a protein, such as ovomucoid or alpha1-acid glycoprotein.

This review article deals with molecularly imprinted polymers (MIPs) as affinity-based separation media for sample preparation. An over view of two types of MIPs (molecularly imprinted particle and monolith) used for the sample preparation and modes of molecularly imprinted SPE (online mode, offline mode, on-column extraction, SPME, and microextraction in packed syringe) is given, focusing on the advantages and disadvantages of these types and modes. Next, problems (template leakage and incompatibility with aqueous conditions) associated with molecularly imprinted SPE and how to overcome those problems are described. Finally, pharmaceutical, food, bioanalytical, and environmental application of molecularly imprinted SPE will be discussed.

Uniform-sized molecularly imprinted polymers (MIPs) for (S)-naproxen and -ibuprofen selectively modified with hydrophilic external layer, restricted access media (RAM)-MIPs, have been prepared. First, the MIP for (S)-naproxen or -ibuprofen was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multistep swelling and thermal polymerization method. Next, a 1:1 mixture of glycerol monomethacrylate and glycerol dimethacrylate was used for hydrophilic surface modification, and it was added directly to the MIP for (S)-naproxen or -ibuprofen 4 h after the start of molecular imprinting. The obtained RAM-MIP material for (S)-naproxen or -ibuprofen was applied for direct serum injection assays of the drug by a column-switching system, consisting of a RAM-MIP material and conventional C18-silica column. However, leakage of the imprint molecule prevented accurate and precise assays of the drug. This problem has been overcome by using the RAM-MIP for (S)-naproxen for the assays of ibuprofen in rat plasma. The optimized column-switching system was applied successfully to the assay of ibuprofen in rat plasma after oral administration.