Development and Validation of a Novel RP-HPLC Analytical Method for Quantification of Amphotericin B

Amphotericin B (AmB) is an antifungal and antiparasitic natural product. AmB is biosynthesized through bacterial fermentation of Streptomyces nodosus. For decades researchers have been investigating new methods to improve the drug formulation. However, drug development research requires validated quantitative analysis techniques to accurately measure drug concentrations. Previously, AmB has been quantified by one of several reverse phase-high performance liquid chromatography (RP-HPLC) that have been reported in literature. However, these methods rely on relatively high disodium EDTA concentration in the mobile phase to separate AmB from its impurities. As a chelating agent, EDTA can bind to metal surfaces within HPLC instrument. Over time this effect can be detrimental, as excess EDTA can precipitate out of solution and negatively impact the integrity of internal instrument components or column performance. Further, reported RP-HPLC methods have relatively high sample run time (> 35 minutes) due to their reliance on isocratic separation. Shorter Run times (< 20 minutes) are desirable for HPLC analytical methods due to the reduced cost mobile phase, as well as to prolong lifetime of detector lamp and stationary phase column. The purpose of this work is to develop and validate a new alternative HPLC method with shorter run times that avoid high EDTA usage. Our developed method achieves a shorter run time by utilizing a gradient HPLC method and avoids the use of EDTA by replacing the agent with 0.2% Acetic Acid.