Analytical and Bioanalytical Chemistry

, Volume 382, Issue 2, pp 290–296 | Cite as

Development and validation of a novel HPLC/ELSD method for the direct determination of tobramycin in pharmaceuticals, plasma, and urine

Special Issue Paper


A novel method for the direct determination of the aminoglycoside tobramycin was developed and validated based on reversed-phase high-performance liquid chromatography (RP-HPLC) with evaporative light scattering detector (ELSD). Using a Waters ODS-2 C18 Spherisorb column with an evaporation temperature of 45°C and nitrogen pressure of 3.5 bar, the selected mobile phase consisted of water/acetonitrile 55:45 containing 1.5 mL L−1 HFBA (11.6 mM) in an isocratic mode at a rate of 1.0 mL min−1. Tobramycin’s retention time was 4.3 min with an asymmetry factor of 1.7. A logarithmic calibration curve was obtained from 1 to 38 μg mL−1 (r > 0.9998). LOD was 0.3 μg mL−1; within-day %RSD was 1.0 (n = 3, 4.7 μg mL−1) and between-day %RSD was 1.1 (3 days within a week). The developed method was applied to the determination of tobramycin in a pharmaceutical crude substance and formulations (eye drops and ointments). Dilution experiments revealed the absence of interference from excipients (no constant and proportional errors); recovery from spiked samples was 99–103% with %RSD < 2.2 (n = 3×3). The developed HPLC/ELSD method was also found to be applicable in the determination of tobramycin in human plasma (0.6–12.5 μg mL−1) and urine (1.5–12.5 μg mL−1) after solid-phase extraction using carboxylate cartridges followed by solvent evaporation (×2 preconcentration). A mean recovery of 86% for plasma and 91% for urine was obtained.


Evaporative light scattering ELSD Tobramycin HPLC Aminoglycosides Pharmaceuticals Plasma Urine 



We gratefully acknowledge financial support from the Greek Ministry of Industry, Energy and Technology, General Secretariat of Research and Technology and the Special Research Account of the University of Athens.


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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  1. 1.Laboratory of Analytical Chemistry, Department of ChemistryUniversity of AthensAthens 15771Greece

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