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

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.

A new simple liquid chromatographic assay for gentamicin in presence of methylparaben and propylparaben

“…Mass spectrometry is the method of choice and offers the required sensitivity and selectivity for confirmation of gentamicin. Several methods based on liquid chromatography coupled to mass spectrometry (LC-MS/MS) are available in literature [20][21][22][23].…”

Exclusive Production of Gentamicin C1a from Micromonospora purpurea by Metabolic Engineering

“…Gentamicin C is produced in a genus of bacteria called Micromonospora [1]. Currently, the engineered strain Micromonospora purpurea Gb1008 series are mainly used in the industrial production of gentamicin C. Gentamicin C mainly consists of five clinically essential components: C1, C1a, C2, C2a, and C2b ( Figure 1) [2]. The five gentamicin components differ from each other mainly at the methylation modification sites.…”

Simultaneous determination of aminoglycoside antibiotics in feeds using high performance liquid chromatography with evaporative light scattering detection

“…Liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis appear to be widespread methods for drugs in complex matrices owing to their high sensitivity and selectivity. [16][17][18][19][20][21] However, the determination of AGs in feed using LC-MS and LC-MS/MS has rarely been reported. Furthermore, an evaporative light scattering detector (ELSD) is a sensitive and universal detector used in conjunction with HPLC and is commonly used for the analysis of compounds such as sugars, lipids and novel antibiotics (no UV absorbance) that cannot be detected by a UV detector.…”