Quantitation of amphotericins by reverse-phase high-performance liquid chromatography

A reverse-phase high-performance liquid chromatographic (HPLC) method was developed for simultaneous potency determination of amphotericin A and amphotericin B in bulk amphotericin B preparations. This single, rapid, specific, and simple method can be used for qualitative and quantitative analyses and is proposed to replace a combination of cumbersome official tests presently in use. This new HPLC method employs an isocratic acidic phosphate bufhotericin A and amphotericin B in bulk amphotericin B preparations. This single, rapid, specific, and simple method can be used for qualitative and quantitative analyses and is proposed to replace a combination of cumbersome official tests presently in use. This new HPLC method employs an isocratic acidic phosphate buffer-methanol mixture in a reverse mode on an octade-cylsilane-bonded silica column at ambient temperature, using UV detection at 313 nm. Results obtained by HPLC compare favorably with the official assay results. During these studies, an apparent heptaenic component, not previously described, was detected in commercial amphotericin B preparations at concentrations ranging from approximately 6 to 14%.