基于UHPLC-Q Exactive Orbitrap HRMS的如意珍宝丸体内外成分分析

Identification of in Vitro and in vivo Chemical Constituents of Ruyi Zhenbaowan Based on UHPLC-Q Exactive Orbitrap HRMS

摘要:

目的 基于超高效液相色谱-四极杆/静电场轨道阱高分辨质谱法（UHPLC-Q Exactive Orbitrap HRMS）对如意珍宝丸所含化学成分及其入血、入脑成分进行定性研究。方法 采用UHPLC-Q Exactive Orbitrap HRMS鉴定如意珍宝丸的化学成分。12只雄性SD大鼠随机分为空白组与如意珍宝丸组，如意珍宝丸组按生药量1.8 g·kg^-1进行如意珍宝丸混悬液灌胃，空白组灌胃等体积纯水。灌胃1.5 h后取血浆与脑脊液，采用ACQUITY UPLC BEH C₁₈色谱柱（2.1 mm×50 mm，1.7 μm），流动相0.1%甲酸水溶液（A）-乙腈（B）梯度洗脱（0~15 min，97%~80%A；15~30 min ，80%~60%A；30~40 min，60%~30%A；40~45 min，30%~5%A），电喷雾离子源（ESI），扫描范围m/z 100~1 500，并结合文献和对照品的相应信息，分别完成在正、负离子模式下对样品进行入血与入脑成分的定性分析。结果 从如意珍宝丸80%甲醇溶液中鉴定出126种化合物，血浆中检测到14种原型成分，脑脊液中检测到7种原型成分，并分析了芹菜素、芹菜素-7-O葡萄糖醛酸苷、高良姜素、甘草苷、胡椒碱、甘草酸、丁香酚、没食子酸和胆酸的裂解规律。结论 该研究实现了如意珍宝丸中多类别体外化学成分、入血及入脑成分的快速表征与鉴定，为该品种药效物质基础与质量控制研究提供了参考。

Abstract:

Objective To identify the chemical constituents of Ruyi Zhenbaowan in vitro and in vivo.Method The chemical constituents of Ruyi Zhenbaowan were identified based on UHPLC-Q Exactive Orbitrap HRMS. A total of 12 male SD rats were randomized into two groups： control （pure water） and Ruyi Zhenbaowan （1.8 g·kg^-1）. The rats were administrated with the suspension of Ruyi Zhenbaowan or pure water by gavage. After 1.5 h， the plasma and cerebrospinal fluid were collected. Chromatographic separation was performed on a Waters ACQUITY UPLC BEH C₁₈ column （2.1 mm × 150 mm， 1.7 μm） with a mixture of 0.1% formic acid aqueous solution （A） and acetonitrile （B） as the mobile phase. Gradient elution was carried out according to the procedure of 0~15 min，97%~80%A；15~30 min ，80%~60%A；30~40 min，60%~30%A；40~45 min，30%~5%A. The ion source was electrospray ionization， and scan range was m/z 100-1 500. The prototype components and the components in the plasma and cerebrospinal fluid were analyzed qualitatively by scanning in positive and negative ion modes and identified by comparison with the data in published literature and the information of standard substances.Result A total of 126 chemical constituents were identified from the 80% methanol solution of Ruyi Zhenbaowan， and 14 and 7 prototype constituents were detected in the plasma and the cerebrospinal fluid， respectively. In addition， the fragmentation rules of apigenin， apigenin-7-O-glucuronide， galangin， liquiritin， piperine， glycyrrhizic acid， eugenol， gallic acid， and cholic acid were deduced.Conclusion This study achieved rapid multicomponent characterization and identification of Ruyi Zhenbaowan in vitro and in vivo， providing theoretical support for exploring active substances and performing quality control.l.