Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, 90610-000 Porto Alegre, RS, Brazil.
Phytochem Anal. 2013 May-Jun;24(3):193-200. doi: 10.1002/pca.2399. Epub 2012 Sep 15.
Achyrobichalcone is a new biflavonoid found in Achyrocline satureioides. It is structurally similar to other bioactive bichalcones that were proven to exert anti-cancer activity. Recently we isolated several achyrobichalcone batches on a semi-preparative scale, showing the need to assess the quality and stability of this substance by analytical methods.
To develop and validate a stability-indicating HPLC method of achyrobichalcone quantification using experimental designs.
The method was developed and optimised by Box-Behnken design using column temperature, flow rate and acetonitrile content in the mobile phase as factors and system suitability parameters as responses. Validation parameters were determined according to official compendiums. Robustness was determined by Plackett-Burman design. Stability of achyrobichalcone was assessed in alkaline, acid, oxidative, thermal and photolytic stress conditions.
The ideal chromatographic conditions were defined from the optimisation: 37 % of acetonitrile, flow rate of 1.2 mL/min and 33°C temperature. All factors were significant for the resolution between achyrobichalcone and impurities peaks and for the retention factor. The mathematical model developed exhibited a good predictive capacity, and the design proved suitable. The HPLC method was successfully validated, being linear, specific, accurate and precise. The robustness test revealed that the flow rate and detection wavelength should be strictly controlled, as they affect achyrobichalcone concentration. The analyte was unstable only in alkaline media.
The new method developed affords evaluation of the quality of achyrobichalcone obtained by isolation, and indicates the stability of the molecule under various stress conditions.
金腰乙素是在 Asteropyrum satureioides 中发现的一种新的二氢查耳酮。它的结构与其他具有生物活性的二氢查耳酮相似，这些二氢查耳酮已被证明具有抗癌活性。最近，我们在半制备规模上分离了几个金腰乙素批次，这表明需要通过分析方法来评估该物质的质量和稳定性。
开发和验证一种使用实验设计的金腰乙素定量的 HPLC 方法，该方法具有稳定性指示性。
通过 Box-Behnken 设计，使用柱温、流动相中的流速和乙腈含量作为因素，以及系统适用性参数作为响应，对方法进行开发和优化。验证参数根据官方药典确定。通过 Plackett-Burman 设计确定稳健性。在碱性、酸性、氧化、热和光解应激条件下评估金腰乙素的稳定性。
从优化中定义了理想的色谱条件：37%的乙腈、流速为 1.2 mL/min 和 33°C 的温度。所有因素对金腰乙素和杂质峰之间的分离度以及保留因子都有重要影响。所开发的数学模型具有良好的预测能力，设计证明是合适的。HPLC 方法成功验证，具有线性、特异性、准确性和精密度。稳健性测试表明，应严格控制流速和检测波长，因为它们会影响金腰乙素的浓度。该分析物仅在碱性介质中不稳定。
开发的新方法可用于评估通过分离获得的金腰乙素的质量，并指示该分子在各种应激条件下的稳定性。
