Simultaneous Quantification of Gentamicin and Colistin Sulfate in Pharmaceuticals using Ion-Pairing and Polarity Gradient Chromatography with Low-UV Detection

For the first time, a simple and rapid method for simultaneous determination of gentamicin sulfate and colistin sulfate in two pharmaceutical formulations for children and adults by ion-pairing reverse phase chromatography and low-UV detection at 215 nm has been developed. This simultaneous analysis is thus a challenge due to the multicomponent mixture of high polar, non volatile and non UV absorbing chromophores. Rapid separation required less than 5 min on a Waters X-Terra^® C18 MS column (50 mm × 4.6 mm i.d., 2.5 μm) with temperature maintained at 35 °C. A linear gradient from 15/85 to 40/60 acetonitrile/water (v/v) with constant hexafluorobutyric acid (HFBA) concentration of 0.05 % (v/v) was used as pairing reagent at 1.5 mL min^?1. In pharmaceutical analysis, the basic and polar compounds are separated by ion-pairing chromatography and the detection of analytes with weak chromophores requires working at low wavelengths. This application is an example of troubleshooting, i.e. baseline drift, due to gradient elution and absorbance of the ion-pairing agent. Baseline drift was minimized by optimizing the HFBA concentration gradient and its slope. Complete analytical validation was carried out according to the International Conference of Harmonization, and real samples were analyzed to demonstrate the applicability of the proposed method for routine use.