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Abstract: A molecularly imprinted solid phase extraction (MISPE) method was developed for the rapid screening of cephalexin in human plasma and serum. The method employed a micro-column packed with molecularly imprinted polymer (MIP) particles for the selective solid phase extraction (SPE) of cephalexin. Since the MIP interacted indiscriminately with two other alpha- aminocephalosporins, cefradine and cefadroxil, their removal was ultimately achieved using differential pulsed elution (DPE) with acetonitrile + 12% acetic acid. Cephalexin was then determined in a final pulsed elution (FPE) with methanol + 1% trifluoroacetic (CF3COOH, TFA) acid. This excellent selectivity represents a significant advance in analytical separation, demonstrating how a MIP can differentiate between molecules that are structurally dissimilar only in their non-hydrogen- bonding moieties, even if their hydrogen-bonding moieties are identical to each other. With UV detection, a concentration detection limit of 0.1 mug/ml (or 2 ng in 20 mul) was afforded for cephalexin. By increasing the CHCl3 flow rate to 1.25 ml/min, each MISPE-DPE-FPE analysis required only 2 min to complete. Rapid screening was demonstrated in a modified MISPE- PE method, which used 14% CH3COOH + CH3CN as the mobile phase, followed by direct PE with 1% TFA + CH3OH. (C) 2003 Elsevier Science B.V. All rights reserved