Determination of gentamicin sulfate and related compounds by high-performance liquid chromatography with evaporative light scattering detection

A rapid and simple method for the separation and quantitation of gentamicin sulfate by HPLC coupled with evaporative light scattering detection (ELSD) has been developed. Detection of the different components of gentamicin is problematic because of the lack of UV absorbing chromophore. The use of the universal ELSD avoids the need for sample derivatization or use of specific detector such as pulsed amperometry. Separation was performed on a highpurity C18 125 mm×4 mm i.d., 3 μm, reversed phase column with 48.5 mM trifluoroacetic acid–methanol (97:3, v/v), as mobile phase at a flow rate of 0.7 ml/min. The influence of the gas nature, gas pressure and temperature of the drift tube of the detector on the detection response was investigated. Optimization was performed with the help of a specific experimental design software. This method allows the determination of the composition in components C1, C1a, C2, C2a and C2b of gentamicin sulfate samples. Mass spectrometry was employed to confirm the ELSD chromatographic profile. The method was validated using methodology described by the International Conference of Harmonization in the field of Medicinal Substances. Commercial samples of different sources were analyzed and results were in good agreement with specifications of both European and United States Pharmacopoeia.