Sensitive fluorimetric determination of gentamicin sulfate in biological matrices using solid-phase extraction, pre-column derivatization with 9-fluorenylmethyl chloroformate and reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of gentamicin in bacterial culture medium or plasma with increased sensitivity and improved separation of the C-1 component. Gentamicin was extracted from the biological matrix with high efficiency using carboxypropyl (CBA)-bonded silica. Derivatization with 9-fluorenylmethyl chloroformate (FMOC-CI) followed by C-18 reversed-phase chromatography allowed the fluorimetric detection of gentamicins C-1, C-1a and C-2. A fourth component, considered to be gentamicin C-2a, was partially resolved from the C-2 peak. Optimal conditions for the extraction and derivatization of gentamicin are described. The detection limit was below 50 mu g/l, the assay was linear to 5 mg/l and showed good reproducibility. It is concluded that pre-column derivatization with FMOC-CI substantially improves the analysis of gentamicin compared with present methods based on reaction with o-phthaldialdehyde.