目的:建立HPLC一测多评法测定蒲地蓝消炎片中多指标成分含量,并验证方法的准确性。方法:应用Box-Behnken响应面法优化样品前处理方法,以料液比、甲醇浓度、提取时间为影响因素,各成分含量综合评分为评价指标,确定最佳提取条件。以黄芩苷为内参物,建立单咖啡酰酒石酸、菊苣酸、千层纸素A-7-O-β-D-葡萄糖醛酸苷、汉黄芩苷、黄芩素的相对校正因子,并计算含量,同时采用外标法测定各成分含量,比较计算值和实测值的差异,以验证一测多评法的准确性和可行性。采用Agilent Poroshell 120 SB-C18色谱柱(50 mm×2.1 mm,2.7 μm),流动相为甲醇-乙腈-0.1%磷酸溶液,梯度洗脱,流速0.3 mL·min-1,柱温30 ℃,检测波长326、274 nm,进样量2 μL。结果:优化后的提取条件为料液比1∶125(0.4 g→50 mL),甲醇浓度60%,提取时间1.5 h。6个成分在各自范围内线性关系良好(r≥0.999 6),平均加样回收率为98.1%~101.8%,RSD为0.34%~1.1%,单咖啡酰酒石酸、菊苣酸、千层纸素A-7-O-β-D-葡萄糖醛酸苷、汉黄芩苷、黄芩素相对校正因子依次为1.008、0.919、1.369、0.857、0.642,且在不同试验条件下重现性良好(RSD<3%),一测多评法计算结果与外标法实测值之间无显著差异。结论:一测多评结合响应面法建立的蒲地蓝消炎片多指标成分含量测定方法准确可靠、简便可行,可用于该制剂质量控制。
Objective: To perform quantitative analysis of multi-target ingredients by single-marker(QAMS)method for Pudilan Xiaoyan tablets, and to validate the accuracy of the method. Methods: With solid-liquid ratio, methanol concentration and extraction time as investigation factors, comprehensive score for the contents of each component as evaluation indicators, the best extraction conditions were optimized by Box-Behnken response surface method. The relative correction factors (RCFs) of caftaric acid, cichoric acid, oroxylin A-7-O-β-D-glucuronide, wogonoside and baicalein were calculated with baicalin as internal reference. Meanwhile, the content of each component was determined by the external standard method (ESM), and the difference between the calculated and measured values were compared to verify the accuracy and feasibility of the method. An HPLC method was applied for quality assessment using an Agilent Poroshell 120 SB-C18 column (50 mm×2.1 mm, 2.7 μm). The mobile phase consisted of methanol-acetonitrile-0.1% phosphoric acid solution (gradient elution) at the flow rate of 0.3 mL·min-1. The column temperature was set at 30 ℃, and the detection wavelengths were 326 nm and 274 nm. The injection volume was 2 μL. Results: The optimal extraction conditions were determined to be 1∶125 for solid-liquid ratio, 60% for methanol concentration, and 1.5 h for extraction time. The linear relationship of the six chemical components was good (r≥0.999 6). The average recoveries were 98.1%-101.8% with RSDs of 0.34%-1.1%. RCFs of caftaric acid, cichoric acid, oroxylin A-7-O-β-D-glucuronide, wogonoside and baicalein to baicalin were 1.008, 0.919, 1.369, 0.857 and 0.642, respectively, and repeatability was good under different experimental conditions (RSD<3.0%). There was no significant difference between the quantitative results of the two methods. Conclusion: The established QAMS method, together with response surface methodology is accurate, reliable, simple and feasible, and can be used for quality control of Pudilan Xiaoyan tablets.
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