Hydrophilic interaction liquid chromatography of aminoglycoside antibiotics with a diol-type stationary phase

Owing to their pronounced polarity, hydrophilic interaction liquid chromatography (HILIC) can be considered as the elective choice for the LC analysis of aminoglycoside (AG) antibiotics. In the present work, a gradient program was optimized for the first time with a diol-type stationary phase and an evaporative light scattering detector (ELSD), thus allowing the almost complete separation of the nine analysed AGs: spectinomycin, dihydrostreptomycin, streptomycin A, gentamicin C1, amikacin, kanamycin A, paromomycin, apramycin and neomycin. In the optimized analysis conditions, analyte retention was found to be governed by a multimodal mechanism encompassing electrostatic, partitioning and hydrophilic interactions. However, the gradient mode of elution complicated a deep understanding of the influence of each contribution on the retention behaviour. The developed HILIC-ELSD method was applied for the analysis of commercial tablets containing neomycin co-formulated with the polypeptide antibiotic bacitracin. The method was fully validated according to the guidelines enshrined in the International Conference on Harmonization (ICH). The use of the diol-type stationary phase was well suited for implementing a successful 2D-HPLC system. Indeed, in order to cope with the absence of chemoselectivity for the couples amikacin/kanamycin and paromomycin/apramycin, a successful 2D-HPLC method was implemented with the “heart-cut” approach and the use of either heptafluorobutyric (for the former) or perfluorooctanoic acid (for the latter) as the ion-pair reagent in the second RP-LC dimension.