Abstract
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.
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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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Megoulas, N.C., Koupparis, M.A. Development and validation of a novel HPLC/ELSD method for the direct determination of tobramycin in pharmaceuticals, plasma, and urine. Anal Bioanal Chem 382, 290–296 (2005). https://doi.org/10.1007/s00216-004-2948-8
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DOI: https://doi.org/10.1007/s00216-004-2948-8