A sensitive non-derivatization method for apramycin and impurities analysis using hydrophilic interaction liquid chromatography and charged aerosol detection
Graphical abstract
A sensitive non-derivatization method was developed for apramycin and impurities using hydrophilic interaction liquid chromatography and charged aerosol detection. More than 16 impurities were separated from apramycin. The current method was compared with BP2010 recommended method. Impurities of apramycin were analyzed by MS. Chromatographic mode, mobile phase and pretreatment were systematically investigated.
Introduction
Apramycin belongs to a class of compounds known as aminoglycoside antibiotics, which are widely used for treating Gram-negative and some Gram-positive bacterial infections [1]. Reliable analytical methods are required for accurate assessment of apramycin purity and minor degradants in routine quality control analysis. However, it is challenge to develop a routinely used HPLC method for quality control of apramycin. Firstly, apramycin and related substances are highly polar compounds with similar structure. They don’t have sufficient retention and adequate resolution in reversed phase liquid chromatography (RPLC). Secondly, apramycin and its impurities lack of a UV-absorbing chromophore [2], [3], [4]. Thirdly, apramycin and related compounds are basic compounds which have tailing and broading peaks [5], [6], [7], [8]. Peak broading and tailing lead to poor resolution and low signal-to-noise ratio. Thus, it is difficult to achieve high sensitivity to detect and estimate low level impurities of apramycin.
Few methods have been reported for the analysis of apramycin and related compounds including post- or pre-column derivatization strong cation exchange-ultraviolet (SCX-UV) methods [9], [10]. Such methods are sensitive for some components of apramycin sample. However, they also have some drawbacks. First, derivatization is tedious and time-consuming. Furthermore, compounds lacking a specific functional group required for derivatization could not be detected. Second, non-volatile buffer solution was used as mobile phase additive for these methods. Non-volatile buffer solution are incompatible with universal detectors, such as evaporative light scattering detector (ELSD) [11], [12]. and charged aerosol detector (CAD) [13], [14]. Recently, reversed phase (RP)-volatile-ion-paring methods were developed to analyze aminoglycoside antibiotics by universal detector including ELSD, CAD and mass spectrometry (MS) [15], [16], [17], [18]. For example, Stypulkowska and co-workers [19]. optimized a non-derivatization method for the analysis of gentamincin using CAD and trifluoroacetic acid (TFA) in the RP mode. This method is simple and it provides high sensitivity and good resolution for gentamincin and related compounds. Nevertheless, it has never been reported for the analysis of apramycin and impurities.
Hydrophilic interaction liquid chromatography (HILIC) is an alternative to RPLC [20]. HILIC provides enough retention and unique selectivity for polar compounds [21], [22]. In our previous study, a cysteine-bonded zwitterionic HILIC stationary phase, Click TE-Cys [23], was introduced for the separation often aminoglycoside antibiotics in one run [24]. Symmetric peak shape and good selectivity were obtained with this type of stationary phase. Furthermore, buffer solutions used with Click TE-Cys, such as ammonium formate, are compatible with CAD and ELSD [21], [23]. To the best of our knowledge, Click TE-Cys column has not yet been utilized for the separation of apramycin and impurities. In this study, a Click TE-Cys based HILIC-CAD method was developed and optimized to achieve good resolution and sensitivity for the analysis of apramycin and related impurities. The effects of sample pretreatment, chromatography mode, and mobile phase composition were investigated. Some of the impurities were identified with MS spectrometric analysis. Furthermore, the presented method was compared with a RP-volatile-ion-paring method and a post-derivatization SCX-UV method recommended by the Chinese Pharmacopoeia (veterinary) 2010 (CP2010) [10].
Section snippets
Apparatus and reagents
The chromatographic system contained a LPG-3400SD pump, a WPS-3000 TSL autosampler, a TCC-3200 column oven and a CAD Veo RS detector. Data were collected and analyzed by Chromeleon version 7.2. The MS data were obtained from a TSQ Quantum mass spectrometer which is a QQQ mass spectrometer. The mass data were collected and analyzed by Xcalibur. All above instruments and workstations were from Thermofisher (Sunnyvale, CA, USA). The post-column derivatization system PCX-2500 was from Pickering
Chromatographic mode and column optimization
Reversed phase chromatography using stationary phases such as C18 is by far the most common mode of liquid chromatography. However, all components of apramycin have weak retentions on C18 column without ion-pairing reagents due to their high polarity. Methods which combine RP column and ion-pairing reagents are usually applied to analyze aminoglycoside antibiotics. However, even using high concentrations ion-pairing reagent HFBA (Fig. 1A). as mobile phase additive, only three peaks with poor
Conclusion
In this study, a sensitive non-derivatization method, HILIC-CAD, was developed for the analysis apramycin and related impurities. Compared to the RP-ion-pairing mode, the HILIC mode provided longer retention and unique selectivity for the analytes of interest. Compared to the method developed on ZIC-pHILIC, the Click TE-Cys based method provided better chromatographic selectivity and peak shape. To eliminate interferences from SO42−, an anion-exhange SPE method based on Dionex II A cartridge
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