Analysis of Spectinomycin Using a pH Stable Specialty Column for Aminoglycoside Antibiotics Separation

Introduction Spectinomycin is a broad-spectrum aminocyclitol antibiotic used to treat bacterial infections in human and animals. Spectinomycin is isolated from the fermentation broth of Streptomyces spectabilis, and it could contain a number of biosynthetically related components including (4S)-dihydrospectinomycin, (4R)-dihydrospectinomycin, dihydroxyspectinomycin, and some degradation products.1 It is important to characterize and quantify the active pharmaceutical ingredient (API) and impurities in the drug product to ensure its quality and safety. As one of the most powerful separation techniques, liquid chromatography is widely used to analyze the aminoglycoside antibiotics quantitatively and qualitatively. Because aminoglycosides and related substances are very hydrophilic and positively charged, it is difficult to retain them on the column when using conventional reversed-phase LC (RPLC). A HILIC approach is also challenging for this analysis due to limited solubility of the aminoglycosides in organic solvents. To address these challenges, ion-pairing reversed phase LC (IP-RPLC) methods have been developed to analyze aminoglycosides using various ion-pairing agents in the mobile phase.2-5 Usually volatile perfluorinated carboxylic acids, such as trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA), and heptafluorobutyric acid (HFBA) are employed to enhance the retention of aminoglycosides on the reversed-phase columns, adjust the selectivity, and, therefore, improve the resolution.