A sensitive non-derivatization method for apramycin and impurities analysis using hydrophilic interaction liquid chromatography and charged aerosol detection

A sensitive non-derivatization method was developed for the analysis of apramycin and impurites using hydrophilic interaction liquid chromatography (HILIC) and charged aerosol detection (CAD). Sample was pretreated with an effective SPE method (recovery >90%) to remove interference with apramycin impurities from sulfate, then analyzed with direct injection. Different chromatography modes of separation and choices of HILIC column were investigated in search of a direct analysis method. The HILIC-CAD method was optimized using a cysteine-bonded zwitterionic HILIC column and compared to the strong cation exchange-ultraviolet (SCX-UV) method with post-column derivatization recommended by the Chinese Pharmacopoeia (veterinary) 2010. The improved chromatographic resolution and peak shape with the HILIC-charged aerosol detection method allows for increase of sample load to 48.9µg from only 2.8µg with the SCX-UV approach. More than 16 impurities were detected with this method with improved resolution, and four were identified with MS, while only 7 impurities were detected with the SCX-UV method. Moreover, the current method has a good precision and reproducibility. The intra-day and inter-day of peak area variability was less than or equal to 4.760% RSD and 9.950%, respectively. The average limit of detection and quantization was 80ng and 200ng injected on the column, respectively. The overall results demonstrated that the presented method can be used as an alternative to SCX-UV method in the analysis of apramycin and impurities. 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. [Display omitted] •A non-derivatization method was optimized for apramycin and related compounds.•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.