基于LC-MS分析4,5-二咖啡酰奎宁酸在大鼠血浆和尿液中的代谢产物

Analysis of metabolites of 4,5-dicaffeoylquinic acid in rat plasma and urine based on LC-MS

应用超高效液相色谱串联高场轨道阱质谱(UPLC-Orbitrap Elite-MS/MS)分析大鼠灌胃给予4,5-二咖啡酰奎宁酸后,血浆和尿液中的代谢物质组。采用Thermo C18色谱柱(2.1 mm×100 mm,1.9μm),以0.1%甲酸水-乙腈为流动相进行梯度洗脱。在负离子模式下采集生物样品的质谱数据,通过比对空白与给药样品,利用Compound Discovery 2.1软件对数据进行提取处理,比对化合物的精确相对分子质量以及质谱碎裂信息,计算二级碎片拟合比,进而归属代谢产物。结果在大鼠血浆中检测到15种代谢产物,在尿液中检测到16种代谢产物。涉及到的代谢反应有甲基化反应、水合反应、去水合反应、还原反应、葡萄糖醛酸化反应、硫酸酯化反应。血和尿中的代谢产物互为补充,初步揭示了此化合物在体内的移行形式和排泄形式。该研究建立起生物样本前处理、高分辨液质联用仪器采集数据、定性软件鉴定代谢产物结构的方法。为之后中药有效成分、体内药效形式的研究打下了基础。

Ultra high performance liquid chromatography tandem high field orbital trap mass spectrometry(UPLC-Orbitrap Elite-MS/MS)method was applied in this paper to analyze the metabolites of 4,5-dicaffeoylquinic acid in rat plasma and urine after oral administration.A gradient elution was performed by using Thermo C18 column(2.1 mm×100 mm,1.9μm),with 0.1%formic acid solution-acetonitrile as the mobile phase.Mass spectral data of biological samples were collected in negative ion mode.The data were extracted by Compound Discovery 2.1 software.Then the blank group samples and the drug samples were compared for exact molecular weight and the mass fragmentation information,and the secondary fragment fitting ratio was calculated to finally attribute the metabolites.As a result,15 metabolites were detected in rat plasma,and 16 metabolites were detected in urine.The involving metabolic reactions included methylation,hydration,dehydration,reduction,glucuronide conjugation,and sulfation reaction.The metabolites in plasma and urine complemented each other and initially revealed the migration and excretion patterns of this compound in the body.A method for pre-processing biological samples,high-resolution LC-MS instrumentation data,and qualitative software was established in this study to identify metabolite structures,laying the foundation for the study of the active ingredients and in vivo pharmacodynamics forms of Chinese medicines.