Enhancement of detection sensitivity and cleanup selectivity for tobramycin through pre-column derivatization

Reversed-phase high-performance liquid chromatography, UV detection and reversed-phase solid-phase extraction (SPE) as analytical methods for pharmaceutical compounds face a challenge when the compound is rather polar and lack UV absorptivity. A good example is tobramycin. To overcome these problems, a method has been developed using pre-column derivatization of tobramycin with o-phthalaldehyde and automated with an autosampler with microrobotic routines. The detection enhancement of the derivatives was achieved by using fluorescence detection which was forty times more sensitive than using UV detection. Recovery studies of standards and spiked serum samples show that pre-SPE derivatization significantly enhances the recoveries (by at least a factor of 3) and the quality of cleanup over post-SPE derivatization.