High-performance liquid chromatographic separation and electrochemical detection of cephalosporins

Pulsed amperometric detection (PAD) is useful for detection of cephalosporins following separation on a C18 column using an acetate buffer solvent with a small percentage of organic modifier. Under these conditions, the indirect PAD mode worked better than direct PAD, with IPAD outperforming both. A gradient program was demonstrated that allowed separation and sensitive electrochemical detection of eleven different cephalosporins with widely differing side chain structures. The cephalosporins could be detected to sub-micromolar levels with this separation. Applications of the method for quantitation of pharmaceutical formulations and for monitoring cephalexin in porcine serum were demonstrated. To improve the detectability of cephalexin, an on-column concentration scheme using separate concentration and elution solvents was applied to porcine serum.