The aim of this study was to determine the content of rutin in Hemidesmus indicus and to optimize the high-performance thin-layer chromatography method. The method was validated in compliance with the International Council for Harmonisation guidelines Q2 (R1) for parameters such as linearity, accuracy, precision, robustness, limit of detection, and limit of quantitation. A Box–Behnken design and response surface methodology has been used to investigate the impact of independent variables on the response. Three independent variables, mobile phase composition (% v/v), mobile phase volume (mL), and duration of saturation (min), were studied. Rutin was verified, and its content was determined using a validated high-performance thin-layer chromatography method with good linearity within the range of 200–1000 ng spot^–1 with r² = 0.9998 and correlation coefficient with calibration curve equation y = 0.0297x + 0.0001. The average percentage recovery values varied from 99.03 to 101.15 and 98.88 to 100.12%, respectively, for in-house and marketed mother tincture). The peak area determination at three different concentration levels shows low values of percentage relative standard deviation (<2%) for inter-day (0.04–0.06) and intra-day (0.04–0.05) precision of rutin. The average content of rutin in extract and marketed mother tincture was 229 ± 0.57 and 210 ± 0.57 μg g^–1. The proposed method was simple, precise, and accurate for the determination of rutin with frequent quality control assessment of H. indicus.

中文翻译：

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Box-Behnken 设计了开发和验证用于估计 Hemidesmus indicus 芦丁的高效薄层色谱方法的途径

本研究的目的是确定Hemidesmus indicus中芦丁的含量。并优化高效薄层色谱法。该方法根据国际协调委员会指南 Q2 (R1) 的线性度、准确度、精密度、稳健性、检测限和定量限等参数进行了验证。Box-Behnken 设计和响应面方法已用于研究自变量对响应的影响。研究了三个自变量，流动相组成 (% v/v)、流动相体积 (mL) 和饱和持续时间 (min)。芦丁经过验证，并使用经过验证的高效薄层色谱法测定其含量，该方法在 200–1000 ng 点^–1范围内具有良好的线性，r ² = 0.9998 和与校准曲线方程y  = 0.0297 x  + 0.0001 的相关系数。对于内部和销售的母酊剂，平均回收率值分别从 99.03 到 101.15 和 98.88 到 100.12% 不等）。三个不同浓度水平的峰面积测定显示芦丁的日间 (0.04-0.06) 和日内 (0.04-0.05) 精度的相对标准偏差百分比较低 (<2%)。提取物和市售母酊中芦丁的平均含量分别为 229 ± 0.57 和 210 ± 0.57 μg g ^-1。所提出的方法简单、精确、准确，可用于芦丁的测定，并经常对H. indicus进行质量控制评估。