Fast determination of underivatized gentamicin C components and impurities by LC-MS using a porous graphitic carbon stationary phase

Gentamicin C antibiotics are important because they are active against many multidrug-resistant Gram-nega- tive bacilli. Unfortunately, their clinical usefulness is limited by their toxicity. Because of the difficulty involved in separat- ing its different components, the US and European pharmaco- peias both specify that the composition of gentamicin C should be determined by liquid chromatography with pulsed electrochemical detection. Here, we assess the usefulness of a porous graphitic carbon (PGC) HPLC column for separating the components of gentamicin C, and report chromatographic conditions that enable its direct characterization by PGC chro- matography directly coupled to electrospray mass spectrome- try. Native major components of gentamicin and impurities in commercial formulations were retained and separated on the PGC column without any need for derivatization, using mo- bile phases basified with ammonium hydroxide. When coupled with detection by conventional electrospray ion trap mass spectrometry (ESI-IT-MS), several previously reported impurities were detected easily, including the most polar gen- tamicin impurity, garamine. When operating in full-scan mode, it was possible to identify and quantitate gentamicin- related compounds using injected samples of only a few pico- grams. Under the described conditions, all analytes were elut- ed in less than 10 min and the LC-MS analyses exhibited excellent stability and linearity. The method’s effectiveness was evaluated by analyzing commercial gentamicin batches and in-house formulations. When the PGC chromatographic system was coupled to an evaporative light-scattering detec- tor, detection limits of 40–70 ng were achieved for various major gentamicin components. The chromatographic method was applied on a semi-preparative scale to purify the five major components.