经典名方泻白散物质基准HPLC指纹图谱的建立及3种指标成分含量测定

Establishment of HPLC fingerprint and determination method of three index components of classical herbal formula substance benchmarks of Xiebai Powder

目的建立经典名方泻白散物质基准HPLC指纹图谱以及3种指标成分桑皮苷A、甘草苷、甘草酸含量测定方法。方法指纹图谱色谱条件:检测波长254 nm/325 nm,柱温35℃;体积流量0.8 mL/min;进样量25μL;流动相为0.1%甲酸水溶液-乙腈,二元梯度洗脱:0~20 min,5%~10%乙腈;20~33 min,10%~15%乙腈;33~50 min,15%~20%乙腈;50~95 min,20%~58%乙腈。采集10批次泻白散物质基准指纹图谱,采用中国药典委员会"中药色谱指纹图谱相似度评价系统2012版"软件对其进行评价。含量测定色谱条件:检测波长237 nm,柱温30℃;体积流量1.0 mL/min;进样量5μL;流动相为0.1%磷酸水溶液-乙腈,二元梯度洗脱:0~10 min,5%~20%乙腈;10~18 min,20%~60%乙腈;18~26 min,60%~100%乙腈;26~38 min,100%乙腈;38~41 min,100%~5%乙腈;41~45 min,5%乙腈。结果根据匹配结果,在254 nm波长下确定了55个共有峰,在325 nm波长下确定了57个共有峰。在共有峰中共指认桑皮苷A(S)、甘草苷、甘草酸铵3种物质。经过方法学研究,其精密度、稳定性、重现性良好。对10批次泻白散物质基准指纹图谱进行评价,结果与对照指纹图谱之间相似度均大于0.9。桑皮苷A、甘草苷、甘草酸铵含量测定方法的平均加样回收率分别为97.82%、97.40%、105.81%,RSD分别为4.41%、2.51%、1.19%,均满足《中国药典》2015年版要求,3种成分分别在25.25~2525、25~2 500、8.5~850 ng线性关系良好,精密度、稳定性、重复性良好。对10批次泻白散物质基准进行含量测定,其中桑皮苷A质量分数为11.6~35.5 mg/g,甘草苷质量分数为0.1~1.6 mg/g,甘草酸质量分数为0.3~2.5 mg/g,3种成分含量差异较大,说明不同产地的桑白皮、甘草药材质量差异较大。结论建立经典名方泻白散物质基准HPLC指纹图谱及多指标成分含量测定的方法,为泻白散物质基准质量标准研究提供依据。

Objective To establish the HPLC fingerprint and the determination method of three index components of the classical herbal formula substance benchmarks of Xiebai Powder. Methods Fingerprint chromatographic conditions were as following: detection wavelength 254 nm/325 nm, column temperature 35 ℃;flow rate 0.8 mL/min;injection volume 25 μL;mobile phase consisting of 0.1% aqueous formic acid(A) and acetonitrile(B);binary gradient elution: 0-20 min, 5%-10% B;20-33 min, 10%-15% B;33-50 min, 15%-20% B;50-95 min, 20%-58% B. Ten batches of substance benchmarks of Xiebai Powder fingerprints were collected and evaluated by the Chinese Pharmacopoeia Committee "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software. Chromatographic conditions of content determination: detection wavelength 237 nm, column temperature 30 ℃;flow rate 1.0 mL/min;injection volume 5 μL;mobile phase consisting of 0.1% aqueous phosphoric acid(A) and acetonitrile(B) for binary gradient elution: 0-10 min, 5%-20% B;10-18 min, 20%-60% B;18-26 min, 60%-100% B;26-38 min, 100% B;38-41 min, 100%-5% B;41-45 min, 5% B. Results Based on the matching results, 55 common peaks were determined at a wavelength of 254 nm, and 57 common peaks were determined at a wavelength of 325 nm. Three substances, mulberroside A(S), liquiritin and ammonium glycyrrhizinate, were identified in the common peaks. After methodological research, its precision, stability and reproducibility were good. Ten batches of substance benchmarks of Xiebai Powder fingerprints were evaluated with reference fingerprints, and their similarities were greater than 0.9. The average recovery rates of mulberroside A(S), liquiritin and ammonium glycyrrhizinate were 97.82%, 97.40% and 105.81%, respectively. The RSD(n = 6) was 4.41%, 2.51% and 1.19%, respectively, which met the require of 2015 edition of the Chinese Pharmacopoeia. The three components had good linearity in the range of 25.25-2525 ng, 25-2 500 ng and 8.5-850 ng, respectively. The method had good precision, stability and repeatability. The contents of 10 batches of substance benchmarks of Xiebai was determined. The content of mulberry A was 11.6-35.5 mg/g, the content of liquiritin was 0.1-1.6 mg/g, and the content of glycyrrhizic acid was 0.3-2.5 mg/g. The range of the contents of these ingredients was large, which indicated that the quality of mulberry husks and licorice herbs from different places was quite different. Conclusion The establishment of the HPLC fingerprint and the determination method of three index components of the classical herbal formula substance benchmarks of Xiebai Powder provided some bases for the study of the quality standard of substance benchmarks of Xiebai Powder.