Utility of Experimental Design in Pre-Column Derivatization for the Analysis of Tobramycin by HPLC－Fluorescence Detection: Application to Ophthalmic Solution and Human Plasma

Asmaa A. El-Zaher ； Marianne A. Mahrouse

《Analytical Chemistry Insights》 2013 (2013/12) Pp. 9-20

https://doi.org/10.4137/ACI.S11612

並列摘要

A novel, selective, and sensitive reversed phase high-performance liquid chromatography (HPLC) method coupled with fluorescence detection has been developed for the determination of tobramycin (TOB) in pure form, in ophthalmic solution and in spiked human plasma. Since TOB lacks UV absorbing chromophores and native fluorescence, pre-column derivatization of TOB was carried out using fluorescamine reagent (0.01%, 1.5 mL) and borate buffer (pH 8.5, 2 mL). Experimental design was applied for optimization of the derivatization step. The resulting highly fluorescent stable derivative was chromatographed on C_(18) column and eluted using methanol:water (60:40, v/v) at a flow rate of 1 mL min^(−1). A fluorescence detector (λ_ex 390 and λ_em 480 nm) was used. The method was linear over the concentration range 20-200 ng mL^(−1). The structure of the fluorescent product was proposed, the method was then validated and applied for the determination of TOB in human plasma. The results were statistically compared with the reference method, revealing no significant difference.

並列關鍵字

tobramycin ； experimental design ； HPLC ； derivatization ； fluorescamine ； plasma