基于UHPLC-LTQ-Orbitrap MS技术分析比较丹酚酸A与丹酚酸B在大鼠体内的代谢过程

Analysis and comparison of metabolic processes of salvianolic acid A and salvianolic acid B in rats based on UHPLC-LTQ-Orbitrap MS technology

采用超高效液相色谱-线性离子阱-静电场轨道阱质谱联用(UHPLC-LTQ-Orbitrap MS)技术分析比较丹酚酸A与丹酚酸B在大鼠体内的代谢产物。大鼠经灌胃给药后,收集不同时间点的血浆及24 h内的尿液,经固相萃取法(SPE)处理后,使用Acquity UPLC BEH C18色谱柱(2.1 mm×100 mm, 1.7μm)和0.1%甲酸水溶液(A)-乙腈(B)流动相系统对其进行梯度洗脱,而后在负离子扫描模式下分析所有大鼠的生物样品。通过获取代谢物的精确相对分子质量与多级质谱信息,结合对照品的特征性裂解规律及文献报道,共鉴定出包括丹酚酸A与丹酚酸B在内的30个代谢产物。其中,来源于丹酚酸A的代谢物有24个,主要代谢途径为酯键断裂、去羟基化、脱羧、氢化、甲基化、羟基化、硫酸酯化、葡萄糖醛酸化以及它们的复合反应;丹酚酸B的代谢产物有15个,主要的生物转化途径为五元环开裂、酯键断裂、脱羧、去羟基化、氢化、甲基化、硫酸酯化、葡萄糖醛酸化以及它们的复合反应。该研究较为完整地阐明了丹酚酸A与丹酚酸B的交叉代谢轮廓,可为其进一步的药效物质基础研究及药理机制挖掘提供借鉴。

The metabolites of salvianolic acid A and salvianolic acid B in rats were analyzed and compared by ultra-high-perfor-mance liquid chromatography with linear ion trap-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS). After the rats were administrated by gavage, plasma at different time points and urine within 24 hours were collected to be treated by solid phase extraction(SPE), then they were gradient eluted by Acquity UPLC BEH C18 column(2.1 mm×100 mm, 1.7 μm) and 0.1% formic acid solution(A)-acetonitrile(B) mobile phase system, and finally all biological samples of rats were analyzed under negative ion scanning mode. By obtaining the accurate relative molecular mass and multi-level mass spectrometry information of metabolites, combined with the characteristic cleavage law of the reference standard and literature reports, a total of 30 metabolites, including salvianolic acid A and B, were identified. Among them, there were 24 metabolites derived from salvianolic acid A, with the main metabolic pathways including ester bond cleavage, dehydroxylation, decarboxylation, hydrogenation, methylation, hydroxylation, sulfonation, glucuronidation, and their multiple reactions. There were 15 metabolites of salvianolic acid B, and the main biotransformation pathways were five-membered ring cracking, ester bond cleavage, decarboxylation, dehydroxylation, hydrogenation, methylation, sulfonation, glucuronidation, and their compound reactions. In this study, the cross-metabolic profile of salvianolic acid A and B was elucidated completely, which would provide reference for further studies on the basis of pharmacodynamic substances and the exploration of pharmacological mechanism.