University Institute of Pharmacy, Kyung Hee University, Seoul, 02447, South Korea.
Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul, 02447, South Korea.
Sci Rep. 2021 Jun 7;11(1):11936. doi: 10.1038/s41598-021-91341-w.
The present study introduces a systematic approach using analytical quality by design (AQbD) methodology for the development of a qualified liquid chromatographic analytical method, which is a challenge in herbal medicinal products due to the intrinsic complex components of botanical sources. The ultra-high-performance liquid chromatography-photodiode array-mass spectrometry (UHPLC-PDA-MS) technique for 11 flavonoids in Genkwa Flos was utilized through the entire analytical processes, from the risk assessment study to the factor screening test, and finally in method optimization employing central composite design (CCD). In this approach, column temperature and mobile solvent slope were found to be critical method parameters (CMPs) and each of the eleven flavonoid peaks' resolution values were used as critical method attributes (CMAs) through data mining conversion formulas. An optimum chromatographic method in the design space was calculated by mathematical and response surface methodology (RSM). The established chromatographic condition is as follows: acetonitrile and 0.1% formic acid gradient elution (0-13 min, 10-45%; 13-13.5 min, 45-100%; 13.5-14 min, 100-10%; 14-15 min, 10% acetonitrile), column temperature 28℃, detection wavelength 335 nm, and flow rate 0.35 mL/min using C (50 × 2.1 mm, 1.7 μm) column. A validation study was also performed successfully for apigenin 7-O-glucuronide, apigenin, and genkwanin. A few important validation results were as follows: linearity over 0.999 coefficient of correlation, detection limit of 2.87-22.41, quantitation limit of 8.70-67.92, relative standard deviation of precision less than 0.22%, and accuracy between 100.13 and 102.49% for apigenin, genkwanin, and apigenin 7-O-glucuronide. In conclusion, the present design-based approach provide a systematic platform that can be effectively applied to ensure pharmaceutically qualified analytical data from complex natural products based botanical drug.
本研究采用分析质量源于设计（AQbD）方法学，为草药药物中具有挑战性的复杂植物源性成分的合格液相色谱分析方法的开发提供了一种系统方法。超高效液相色谱-光电二极管阵列-质谱（UHPLC-PDA-MS）技术用于金莲花中的 11 种黄酮类化合物，贯穿整个分析过程，从风险评估研究到因子筛选试验，最后通过中心组合设计（CCD）进行方法优化。在这种方法中，柱温与流动相斜率被确定为关键方法参数（CMP），并通过数据挖掘转换公式将 11 种黄酮类化合物峰的分辨率值均用作关键方法属性（CMA）。通过数学和响应面法（RSM）计算设计空间中的最佳色谱方法。建立的色谱条件如下：乙腈-0.1%甲酸梯度洗脱（0-13min，10-45%；13-13.5min，45-100%；13.5-14min，100-10%；14-15min，10%乙腈），柱温 28℃，检测波长 335nm，流速 0.35mL/min，采用 C（50×2.1mm，1.7μm）柱。还成功地对芹黄素 7-O-葡萄糖醛酸苷、芹黄素和金莲花黄素进行了验证研究。一些重要的验证结果如下：线性相关系数大于 0.999，检测限为 2.87-22.41，定量限为 8.70-67.92，精密度的相对标准偏差小于 0.22%，芹黄素、金莲花黄素和芹黄素 7-O-葡萄糖醛酸苷的准确度在 100.13%至 102.49%之间。总之，本设计方法为基于植物的草药复杂天然产物提供了一个系统的平台，可以有效地应用于确保合格的药学分析数据。
