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

Abstract

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.

Do Ophthalmic Solutions of Amphotericin B Solubilised in 2-Hydroxypropyl-γ-Cyclodextrins Possess an Extended Physicochemical Stability?

“…The results that were obtained were coherent with those published initially by Chang et al in terms of retention times and relative retention times, and breakdown studies allowed the detection of multiple compounds, all correctly separated from the main amphotericin B peak. They are also in accordance with the results of a very recent study by Montenegro et al who also developed a stability indicating liquid chromatography–diode array detector method for amphotericin B quantification [ 37 ]. They confirmed that amphotericin B degrades rapidly under acidic, alkaline, oxidative and radiation exposure conditions, detecting 16 breakdown products, and made an interesting tentative shot at identifying some of the compounds using direct injection electrospray ionization mass spectrometry and electrospray ionization tandem mass spectrometry.…”

AmBisome® Formulations for Pediatrics: Stability, Cytotoxicity, and Cost-Effectiveness Studies

“…It has been reported that AmB suffers from degradation when exposed to light, heat, radical initiators, and extreme pH conditions. The by-products have not been isolated yet, but the most probable reactions involve the highly conjugated polyene backbone, similar to a lipid oxidation [ 27 ]. Moreover, previous studies suggested that the degradation products may increase drug toxicity, such as acute hemolysis [ 28 ].…”

“…The chromatographic conditions used in this study were based on those reported by Espada et al, with some changes [ 29 ]. The detection wavelength for AmB and its related impurities, eventually present during in-use conditions storage, was set at 406 nm, as already proposed for the detection of some degradation products [ 27 ]. Linearity was maintained with stock solutions in methanol ranging from 38 to 360 µg/mL AmB.…”

Sixty years of Amphotericin B: An Overview of the Main Antifungal Agent Used to Treat Invasive Fungal Infections

“…A recent study showed insignificant degradation of AMB in the presence of water [230]; this fact could be related to AMB's low solubility in aqueous and neutral pH vehicles. Exposure of AMB to ± 70°C for up to 7 days did not provoke thermal degradation.…”