Determination of Gentamicins C1, C1a, and C2 in Plasma and Urine by HPLC

Abstract

Gentamicin is an aminoglycoside antibiotic complex containing gentamicins C(1), C(1a), and C(2). Few methods have been described for analysis of the three gentamicin components separately in biological fluids, and none has been used in pharmacokinetic studies. Determination of the three gentamicins separately may have pharmacokinetic and toxicological implications. The present study describes development of an HPLC method for the analysis of gentamicin C(1), C(1a), and C(2) components in plasma and urine. The three components were isolated by preparative chromatography and their identities verified by thin-layer chromatography, HPLC, mass spectrometry, nuclear magnetic resonance spectroscopy, and melting point determination. The gentamicins were extracted from the biological matrix by use of Tris buffer and polymer phase solid-phase extraction. Derivatization was carried out in the solid-phase extraction cartridge with 1-fluoro-2, 4-dinitrobenzene. The 2,4-dinitrophenyl derivatives were separated with reversed-phase HPLC and quantified by the ultraviolet absorbance at 365 nm. 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 urine. The method was validated for human and dog plasma and urine. The method was repeatable and enabled the analysis of gentamicins C(1), C(1a), and C(2) in plasma and urine in concentrations covering the therapeutic range of the drug, thus being suitable for therapeutic drug monitoring and pharmacokinetic studies.