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

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Abstract

Gentamicin C antibiotics are important because they are active against many multidrug-resistant Gram-negative bacilli. Unfortunately, their clinical usefulness is limited by their toxicity. Because of the difficulty involved in separating its different components, the US and European pharmacopeias 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 chromatography directly coupled to electrospray mass spectrometry. Native major components of gentamicin and impurities in commercial formulations were retained and separated on the PGC column without any need for derivatization, using mobile 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 gentamicin 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 picograms. Under the described conditions, all analytes were eluted 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 detector, 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.

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Acknowledgments

This study was conducted within the PRIN Project “Design and development of microsystems (gel-beads) for drug delivery by laminar jet break-up and dielectric treatments” financed by MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca). We also received financial support from the Campania regional government under the project POR Campania FSE 2007/2013. 4-17-1; CUP B25B09000000007 (STRAIN).

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  1. Dipartimento di Farmacia, Università degli Studi di Salerno, Via Giovanni Paolo II 132, 84084, Fisciano, Salerno, Italy

    Manuela Rodriquez, Daniel S. Cretoso, Maria Anna Euterpio, Paola Russo, Carlo Crescenzi & Rita P. Aquino

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  1. Manuela Rodriquez
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  2. Daniel S. Cretoso
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  3. Maria Anna Euterpio
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  4. Paola Russo
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  5. Carlo Crescenzi
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  6. Rita P. Aquino
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Correspondence to Carlo Crescenzi.

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Rodriquez, M., Cretoso, D.S., Euterpio, M.A. et al. Fast determination of underivatized gentamicin C components and impurities by LC-MS using a porous graphitic carbon stationary phase. Anal Bioanal Chem 407, 7691–7701 (2015). https://doi.org/10.1007/s00216-015-8933-6

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  • DOI: https://doi.org/10.1007/s00216-015-8933-6

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