HPLC-UV/ESI-MS法分析丹参不同提取组分

4 components from Salviae Miltiorrhizae extracted by HPLC-UV/ESI-MS

目的对丹参4种不同组分进行HPLC-UV-MS分析，获得各组分的HPLC特征图谱并对各特征峰进行初步MS鉴定，同时定量测定指标成分丹参素、丹酚酸B、丹参酮ⅡA和隐丹参酮的含量，以便更深入的探讨其在抗动脉粥样硬化作用的物质基础及谱效关系。方法采用Hedera ODS-2色谱柱（4．6min×250mm，5μm），0．5％甲酸水溶液-乙腈（0min，80：20；50min，15：85）梯度洗脱，流速：1．0mL·min^-1，检测波长：280nm，建立以上各组分的HPCL指纹图谱，同时对各特征峰进行离子范围为150-750正负源ESI-MS总离子扫描，对相应特征峰的特征离子进行鉴定，用分子离子检测模式（SIR）分别对丹参素、丹酚酸B、丹参酮ⅡA和隐丹参酮进行定量测定。结果所得HPLC特征图的特征峰峰形尖锐、对称，均达到基线分离；14个特征峰经质谱鉴定，对照有关文献可初步确定其成分；丹参素、丹酚酸B、丹参酮ⅡA和隐丹参酮分别在0．05-10．0、0．05-20．0、0．05-20、0．05-20μg·mL^-1与峰面积呈良好的线性关系（r≥0．9998），平均加样回收率分别为98．1％、99．0％、98．9％、99．5％。结论所得4种组分的HPLC特征图谱适合下一步的译效学及相关药理活性成分筛选研究；定量测定方法准确可靠，适合丹参药材中上述成分的含量测定；所建立的HPLC特征图谱能为丹参药材指纹图谱的建立提供依据。

Objective To establish the HPLC fingerprint of 4 components from Radix Salviae Miltiorrhizae （RSM） extract, to qualitatively analyze the major marker peaks in the HPLC profile by MS, and to simultaneouly and quantitatively determine danshensu, salvianolic acid B, cryptotansbinone, and tanshinone Ⅱ A, and further study the relationship between their fingerprint and pharmacological effect. Methods HPLC analysis was carried out on an ODS-2 column with linear gradient elution. The mobile phases were composed of formic acid aqueous solution （0. 5%） （A） and acetonitrile （B）. The gradient was as follows： 0 min, 80X A, 20% B; 50 min, 15% A, 85%, B, strarting with 20% B, and injection volume of 20μL. The fingerprint profiles were recorded at an optimized wavelength of 280 nm. And 0.2 mL · min^-1 flow phase was transferred into the MS system, and 150-750 total positive and negative irons were scanned to qualitatively analyze the marker peaks in HPLC profile on line. Molecular ions were monitored to quantitatively determine danshensu, salvianolic acid B, cryptotanshinone, and tanshinone, respectively. Results Most of the the characterized peaks in the HPLC fingerprint were well separated and provided much chemical information of the pharmacological compounds, and 14 marker peaks were qualitatively determined. The quantitative method was valid, and the linear range of danshensu, salvianolic acid B, cryptotanshinone, and tanshinone, Ⅱ A was 0. 05-10. 0, 0. 05-20. 0, 0. 05420, and 0. 05-20μg · mL^-1 with good correlation coefficient （r≥0. 999 8）. The mean recovery rates from fortified samples （n= 5） of dihydrotanshinone Ⅰ , cryptotanshinone, tanshinone Ⅰ , and tanshinone Ⅱ A were 98.1%, 99.0%, 98. 9%, and 99.5%, respectively, with variation coefficient RSD≤1. 5%. Conclusion The established fingerprints of 4 components from SM extract are suitable for the screening of pharmacological active constituents and effect-fingerprint study. The quantitative method is accurate, simple and suitable for the determination of the above 4 components in crude herbal of SM.