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Abstract: A restricted access media-molecularly imprinted polymer (RAM- MIP) for propranolol (PRP) has been prepared for direct injection analysis of beta-blockers in biological fluids. First, the MIP for PRP was prepared using methacrylic acid and ethylene glycol dimethacrylate as the functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method. Next, a 1: 1 mixture of glycerol monomethacrylate and glycerol dimethacrylate was used for hydrophilic surface modification, and added directly to the MIP for PRP after 4 h from the start of polymerization. Then further polymerization was carried out for 20 h. The obtained RAM-MIP for PRP showed excellent molecular recognition ability for PRP, good ones for alprenolol ( ALP) and pindolol, and fair ones for other beta-blockers. The RAM-MIP was applied for direct injection analysis of ALP enantiomers in a rat plasma sample by a column-switching HPLC system using a beta- cyclodextrin phenylcarbamate-bonded silica column as the analytical column. The calibration graph, constructed from peak area versus each ALP enantiomer concentration, was linear with a correlation coefficient of > 0.999 over the concentration ranges of 12.5 - 250 ng ml(-1). The limit of quantitation was 12.5 ng ml(-1) with a 50 mul injection. This method could be applicable for the assay of ALP enantiomers at the therapeutic plasma levels, and have wide applicability for the assay of beta-blockers in biological fluids