Determination of Gentamicin C-subtypes in Inner Ear Perilymph Using Liquid Chromatography with Fluorescence Detection

Gentamicin is a broad-spectrum aminoglycoside used frequently to treat gram-positive and gram-negative bacterial infections. In this study, a new, simple, fast, and sensitive isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method was developed and validated for the detection and quantification of fluorescent OPA-ethanethiol derivatized gentamicin in very small biological sample volumes. To our knowledge, there is no report of the use of ethanethiol for the derivatization of gentamicin with OPA, and the simultaneous determination of the four major C-subtypes of gentamicin using OPA-derivatives. Optimum chromatographic conditions were achieved on a C₁₈ column with a mobile phase consisting of methanol, glacial acetic acid, and an aqueous solution of sodium 1-heptanesulfonate at a flow rate of 1.0 mL/min under ambient conditions. The method was successfully validated according to the acceptance criteria of USP guidelines in terms of selectivity, linearity, accuracy, precision, and sensitivity. The linearity of the method was demonstrated with a concentration range of gentamicin (10-400 ng/mL) prepared in artificial perilymph. The limit of detection was 0.2 ng/mL and the limit of quantification was 10-11 ng/mL for all four major C-subtypes of gentamicin. Finally, due to its high sensitivity, this method was successfully applied to quantify gentamicin concentrations in the small volumes of perilymph present in the inner ear of mice. Thus, this RP-HPLC-fluorescence method for detecting derivatized gentamicin in preclinical models is promising in terms of simplicity and high sensitivity.