Determination of gentamicins C-I, C-Ia, and C-2 in plasma and urine by HPLC

Background: Gentamicin is an aminoglycoside antibiotic complex containing g entamicins C-1, C-1a, and C-2. Few methods have been described for analysis of the three gentamicin components separately in biological fluids, and no ne has been used in pharmacokinetic studies. Determination of the three gen tamicins separately may have pharmacokinetic and toxicological implications . The present study describes development of an HPLC method for the analysi s of gentamicin C-1, C-1a, and C-2 components in plasma and urine. Methods: The three components were isolated by preparative chromatography a nd their identities verified by thin-layer chromatography, HPLC, mass spect rometry, nuclear magnetic resonance spectroscopy, and melting point determi nation. The gentamicins were extracted from the biological matrix by use of Tris buffer and polymer phase solid-phase extraction. Derivatization was c arried out in the solid-phase extraction cartridge with 1-fluoro-2,4-dinitr obenzene. The 2,4 dinitrophenyl derivatives were separated with reversed-ph ase HPLC and quantified by the ultraviolet absorbance at 365 nm. Results: The detector response was linear from the limit of quantification to 50 mg/L for the individual components. The limit of quantification was 0 .07 mg/L for gentamicin C-1 and 0.1 mg/L for gentamicins C-2 and C-1a. The recovery of the gentamicin components was 72% from plasma and 98% from urin e. The method was validated for human and dog plasma and urine. Conclusions: The method was repeatable and enabled the analysis of gentamic ins C-1 C-1a, and C-2 in plasma and urine in concentrations covering the th erapeutic range of the drug, thus being suitable for therapeutic drug monit oring and pharmacokinetic studies. (C) 2000 American Association for Clinic al Chemistry.