Non-derivatization approach to high-performance liquid chromatography-fluorescence detection for aminoglycoside antibiotics based on a ligand displacement reaction.

An indirect fluorescence detection method has been developed for detecting the aminoglycoside antibiotics following chromatographic separation. This approach to detection is based on a displacement reaction between the aminoglycosides and a copper(II)-L-tryptophan (L-Trp) complex, Cu(L-Trp)2. The aminoglycosides, which contain multiple amino groups, have strong affinities for the Cu(II) ion and displace L-Trp from the Cu(L-Trp)2 complex. The resulting increase in L-Trp fluorescence, which is quenched when coordinated to Cu(II), is indicative of the presence of the aminoglycoside. Fluorescence titration data indicate that there is a stoichiometric ratio of 1:1 between the reaction of the aminoglycosides with Cu(L-Trp)2. This HPLC detection scheme is implemented postcolumn by mixing a buffered Cu(L-Trp)2 solution with the column eluent prior to detection. The aminoglycosides were separated with the use of a column packed with a polymeric strong cation-exchanger. Separation and detection variables were optimized and are discussed. The detection limits for the aminoglycosides tested ranged from 4.2 to 14.5 ng injected (S/N=3). A linear working curve was achieved for amikacin in the range of 29-586 ng for a six point linearity test. The developed separation and detection scheme was further tested by analyzing commercial pharmaceutical formulations of these antibiotics.