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

Area of Science:

• Analytical Chemistry
• Pharmaceutical Analysis

Background:

• Traditional methods for analyzing amphotericin B preparations are cumbersome.
• Simultaneous determination of amphotericin A and B is crucial for quality control.

Purpose of the Study:

• To develop and validate a single, rapid, and specific high-performance liquid chromatography (HPLC) method for the simultaneous potency determination of amphotericin A and B.
• To propose this HPLC method as a replacement for existing official tests.

Main Methods:

• Development of a reverse-phase HPLC method using an isocratic acidic phosphate buffer-methanol mixture.
• Utilized an octadecylsilane-bonded silica column at ambient temperature with UV detection at 313 nm.
• Method validated for qualitative and quantitative analyses.

Main Results:

• The developed HPLC method provides simultaneous potency determination of amphotericin A and B.
• Results from the HPLC method favorably compare with official assay results.
• A previously undescribed heptaenic component was detected in commercial amphotericin B at concentrations of 6–14%.

Conclusions:

• The new HPLC method is a suitable replacement for current official tests due to its speed, simplicity, and specificity.
• This method allows for accurate qualitative and quantitative analysis of amphotericin A and B.
• The identification of the heptaenic component necessitates further investigation into amphotericin B preparations.