Advanced green chemistry, HPLC method development and validation: Integrating stability indicating, quality by design (QbD), in ferulic acid and its application in NLCs-based nanoformulation

The biopharmaceutical class IV classification of ferulic acid (FA) restricts its use. FA-loaded nanostructured lipid carriers (NLCs) were developed to overcome these issues. It is difficult to quantify the lowest concentration of the drug by another method. The proposed method can determine the lowest concentration in nanoformulation and biological fluids including urine, plasma, blood, and tissues. The proposed method utilizes a novel QbD-based approach, which reduces trial-and-error production costs and market recalls and maintains a risk-benefit ratio, due to the use of optimized inputs and outputs. The use of a green chemistry approach (Acetonitrile: water) can detect even small degradants. The degradant found under stress conditions ensures the safety, efficacy, quality, and stability of the formulations. The optimized method showed linearity with r² 0.9988 in a range of 10-60 µg/ml at a retention time of 3.690±0.01 at a run time of 8 minutes. The recovery rate of FA was found in a range of 95.28±0.54% to 97.98±0.48%. The obtained %RSD precision was (0.01-0.21%) for intra-day, whereas inter-day precision was (0.09-0.51%). The lowest concentrations to be detected and quantified were 2.4 and 7.3µg/ml. The AQbD approach built an optimized method providing significant output. The found stress degradation recovery was (6.2±0.98% to 33.46±0.22%), thus from the recovered degradant concentrations, further work for safety, efficacy, quality, and stability can proceed. Thus RP-HPLC adheres to ICH guidelines and is found to be straightforward, precise, and sensitive for routine quality control analysis of FA in bulk and nanoformulations, plasma, tissues, and other biological fluids.

阿魏酸（ferulic acid, FA）的生物药剂学IV级分类限制了其临床应用。为此，本研究开发了负载阿魏酸的纳米结构脂质载体（nanostructured lipid carriers, NLCs）以克服上述局限。传统检测方法难以定量该药物的最低浓度，而本研究提出的方法可在纳米制剂以及尿液、血浆、全血、组织等生物体液中实现最低药物浓度的定量检测。该方法采用基于质量源于设计（Quality by Design, QbD）的全新策略，通过优化输入与输出参数，降低了试错生产成本与产品市场召回风险，同时维持了合理的风险收益比。本方法采用乙腈-水体系的绿色化学检测策略，甚至可检出痕量降解产物；通过胁迫条件下检出的降解产物，可有效保障制剂的安全性、有效性、质量与稳定性。优化后的检测方法在10~60 μg/mL浓度范围内呈现良好线性关系，决定系数r²为0.9988，保留时间为3.690±0.01 min，总运行时长为8分钟。阿魏酸的加样回收率介于95.28±0.54%至97.98±0.48%之间。日内精密度的相对标准偏差（relative standard deviation, %RSD）为0.01%~0.21%，日间精密度的相对标准偏差则为0.09%~0.51%。本方法的最低检测浓度与定量浓度分别为2.4 μg/mL与7.3 μg/mL。分析质量源于设计（Analytical Quality by Design, AQbD）策略构建的优化方法可获得优异的检测性能。胁迫条件下的降解产物回收率介于6.2±0.98%至33.46±0.22%之间，基于上述降解产物浓度数据，可进一步开展制剂安全性、有效性、质量与稳定性相关研究。综上，本反相高效液相色谱（Reversed-Phase High Performance Liquid Chromatography, RP-HPLC）方法符合人用药品注册技术要求国际协调会（International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, ICH）指南要求，可简便、精准、灵敏地用于原料药、纳米制剂、血浆、组织及其他生物体液中阿魏酸的常规质量控制分析。