一种快速、稳定性指示的、熔芯 HPLC 方法，用于同时测定抗疟复方固定剂量组合产品中的β-青蒿素和盐酸阿莫地喹。
A rapid stability-indicating, fused-core HPLC method for simultaneous determination of β-artemether and lumefantrine in anti-malarial fixed dose combination products.
机构信息
Drug Quality and Registration (DruQuaR) group, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, Ghent, B-9000, Belgium.
出版信息
Malar J. 2013 Apr 30;12:145. doi: 10.1186/1475-2875-12-145.
BACKGROUND
Artemisinin-based fixed dose combination (FDC) products are recommended by World Health Organization (WHO) as a first-line treatment. However, the current artemisinin FDC products, such as β-artemether and lumefantrine, are inherently unstable and require controlled distribution and storage conditions, which are not always available in resource-limited settings. Moreover, quality control is hampered by lack of suitable analytical methods. Thus, there is a need for a rapid and simple, but stability-indicating method for the simultaneous assay of β-artemether and lumefantrine FDC products.
METHODS
Three reversed-phase fused-core HPLC columns (Halo RP-Amide, Halo C18 and Halo Phenyl-hexyl), all thermostated at 30°C, were evaluated. β-Artemether and lumefantrine (unstressed and stressed), and reference-related impurities were injected and chromatographic parameters were assessed. Optimal chromatographic parameters were obtained using Halo RP-Amide column and an isocratic mobile phase composed of acetonitrile and 1 mM phosphate buffer pH 3.0 (52:48; V/V) at a flow of 1.0 ml/min and 3 μl injection volume. Quantification was performed at 210 nm and 335 nm for β-artemether and for lumefantrine, respectively. In-silico toxicological evaluation of the related impurities was made using Derek Nexus v2.0®.
RESULTS
Both β-artemether and lumefantrine were separated from each other as well as from the specified and unspecified related impurities including degradants. A complete chromatographic run only took four minutes. Evaluation of the method, including a Plackett-Burman robustness verification within analytical QbD-principles, and real-life samples showed the method is suitable for quantitative assay purposes of both active pharmaceutical ingredients, with a mean recovery relative standard deviation (± RSD) of 99.7 % (± 0.7%) for β-artemether and 99.7 % (± 0.6%) for lumefantrine. All identified β-artemether-related impurities were predicted in Derek Nexus v2.0® to have toxicity risks similar to β-artemether active pharmaceutical ingredient (API) itself.
CONCLUSIONS
A rapid, robust, precise and accurate stability-indicating, quantitative fused-core isocratic HPLC method was developed for simultaneous assay of β-artemether and lumefantrine. This method can be applied in the routine regulatory quality control of FDC products. The in-silico toxicological investigation using Derek Nexus® indicated that the overall toxicity risk for β-artemether-related impurities is comparable to that of β-artemether API.
背景
世界卫生组织（WHO）推荐青蒿素类固定剂量复方（FDC）产品作为一线治疗药物。然而，目前的青蒿素类 FDC 产品，如β-青蒿素和本芴醇，本身不稳定，需要控制分发和储存条件，而在资源有限的环境中，这些条件并不总是具备。此外，质量控制受到缺乏合适分析方法的阻碍。因此，需要一种快速、简单但能指示稳定性的方法，用于同时测定β-青蒿素和本芴醇 FDC 产品。
方法
评估了三种反相融合核 HPLC 柱（Halo RP-Amide、Halo C18 和 Halo Phenyl-hexyl），均在 30°C 下恒温。注入β-青蒿素和本芴醇（未受应力和受应力）以及参考相关杂质，并评估色谱参数。使用 Halo RP-Amide 柱和等度流动相（乙腈和 1mM 磷酸盐缓冲液 pH3.0（52:48；V/V），流速为 1.0ml/min 和 3μl 进样体积，获得最佳色谱参数。β-青蒿素在 210nm 处，本芴醇在 335nm 处进行定量。使用 Derek Nexus v2.0®对相关杂质进行了虚拟毒理学评估。
结果
β-青蒿素和本芴醇彼此以及与指定和非指定相关杂质（包括降解产物）均得到了分离。整个色谱运行只需四分钟。方法评估包括基于分析 QbD 原则的 Plackett-Burman 稳健性验证，以及实际样品表明，该方法适用于定量测定两种活性药物成分，β-青蒿素的平均回收率相对标准偏差（±RSD）为 99.7%（±0.7%），本芴醇的平均回收率相对标准偏差（±RSD）为 99.7%（±0.6%）。Derek Nexus v2.0®预测所有鉴定出的β-青蒿素相关杂质的毒性风险与β-青蒿素原料药（API）本身相似。
结论
开发了一种快速、稳健、精确、准确的指示稳定性、定量融合核等度 HPLC 方法，用于同时测定β-青蒿素和本芴醇。该方法可应用于 FDC 产品的常规监管质量控制。使用 Derek Nexus®进行的虚拟毒理学研究表明，β-青蒿素相关杂质的整体毒性风险与β-青蒿素 API 相当。
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