Methodology Development and Validation of Amphotericin B Stability by HPLC‑DAD

• Published in: Journal of the Brazilian Chemical Society Vol. 31; no. 5; pp. 916 - 926
• Main Authors: Montenegro, Marciela, de Souza, Stefânia, Leão, Raquel, Rocha, Helvécio, de Rezende, Claudia, de Souza, Rodrigo
• Format: Journal Article
• Language: English
• Published: Sociedade Brasileira de Química 01-05-2020
• Subjects: CHEMISTRY, MULTIDISCIPLINARY; forced degradation; amphotericin B; HPLC-DAD; degradation products; indicative stability method
• ISSN: 0103-5053; 1678-4790
• DOI: 10.21577/0103-5053.20190256

The high worldwide consumption of antibiotics and their complex impurity profiles has drawn the attention of the scientific community to the development and validation of stability methods for these drugs. Amphotericin B is an antibiotic that is a natural fermentation product of bacterium Streptomyces nodosus, used as a broad-spectrum antifungal agent, and is highly unstable. For this reason, the objective of this work is the development and validation of an indicative method of stability by high performance liquid chromatography (HPLC) associated to diode array detector (DAD) for amphotericin B (AMB). To achieve this, the chromatographic profiles of acid, basic, oxidative and thermal degradation caused by AMB exposure to water and light were verified by HPLC-DAD, using an isocratic method under the following conditions: C18 chromatographic column (200 × 4.6 mm-5 µm), mobile phase composed of 65:35 of organic phase (methanol/acetonitrile in 41:18)/aqueous phase (2.5 mmol L-1 of disodium edetate, pH 5.0), flow rate of 1.0 mL min-1, injection volume of 20 µL, column temperature 30 ± 2 °C and wavelength of 383 nm. After identification of these profiles, the method was validated according to recommendations of the International Conference on Harmonization guidelines, and then, the active pharmaceutical ingredient (API) peak purity grade, percentage of drug degradation, occurrence of impurities peak and its identification by mass spectrometry (MS) with electrospray ionization (ESI), under positive ionization mode, were evaluated. It is suggested that the main degradation products were amphotericin B (2), degradation product 1 (DP1) and degradation product 2 (DP2) in acid and oxidative medium. Amphotericin B was stable in the presence of water and at 70 ± 2 °C for seven days.