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

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.

全球范围内抗生素的高消费量及其复杂的杂质谱，已引起科学界对这类药物稳定性研究方法开发与验证的广泛关注。
两性霉素B（Amphotericin B, AMB）是由结节链霉菌（Streptomyces nodosus）发酵产生的天然抗生素，作为广谱抗真菌药物被广泛使用，但其稳定性极差。
鉴于此，本研究旨在开发并验证一种基于高效液相色谱-二极管阵列检测器（High Performance Liquid Chromatography, HPLC；Diode Array Detector, DAD）的两性霉素B稳定性指示性分析方法。
为实现该研究目标，本研究采用等度洗脱方法，通过HPLC-DAD分析了两性霉素B在水、光照条件下引发的酸解、碱解、氧化及热降解的色谱行为，色谱条件如下：采用规格为200×4.6 mm、5 μm的C18色谱柱；流动相由有机相（甲醇/乙腈体积比41:18）与水相（2.5 mmol·L⁻¹乙二胺四乙酸二钠，pH 5.0）按65:35的体积比混合而成；流速为1.0 mL·min⁻¹；进样体积20 μL；柱温设置为30±2℃；检测波长383 nm。
明确上述降解色谱行为后，本方法依据国际人用药品注册技术协调会（International Conference on Harmonization, ICH）指导原则完成验证，并对活性药物成分（Active Pharmaceutical Ingredient, API）的峰纯度、药物降解百分比、杂质峰的出现情况，以及采用电喷雾电离（Electrospray Ionization, ESI）正离子模式的质谱（Mass Spectrometry, MS）对杂质进行鉴定。
研究表明，在酸性及氧化介质中，主要降解产物为两性霉素B（2）、降解产物1（DP1）与降解产物2（DP2）；
两性霉素B在水溶液中及70±2℃条件下放置7天后仍保持稳定。