基于UPLC-Q/TOF-MS的甘草酸单铵和甘草酸二铵在大鼠体内移行成分表征

Characterization of transitional components of monoammonium glycyrrhizinate and diammonium glycyrrhizinate in rats based on UPLC-Q/TOF-MS

目的采用超高效液相色谱-四极杆/飞行时间高分辨质谱技术(UPLC-Q/TOF-MS)对甘草酸单铵和甘草酸二铵在大鼠体内的代谢轮廓进行表征,探讨两者可能的代谢途径。方法大鼠分别灌胃给予甘草酸单铵和甘草酸二铵后,收集不同时间点大鼠脑组织、血浆、尿液、粪样等生物样本。经沉淀蛋白后,采用UPLC-Q/TOF-MS技术,以含0.1%甲酸的水-乙腈为流动相进行梯度洗脱;在正离子模式下采集生物样本的质谱数据。采用质量残差亏损过滤技术并结合原型成分的质谱裂解规律,预测代谢物类型,对两者相关的体内代谢产物进行鉴定。结果甘草酸单铵和甘草酸二铵在大鼠体内主要以代谢物形式存在,共鉴定得到20个代谢产物,两者主要通过肠道排泄,在体内原型化合物脱糖基形成的甘草次酸基础上发生一系列的Ⅰ相氧化还原反应是主要代谢类型;甘草次酸是在生物样本中主要暴露成分,提示了其可能是甘草酸单铵及甘草酸二铵在体内发挥药效的关键药效成分。结论首次阐明了甘草酸单铵及甘草酸二铵在大鼠体内的代谢轮廓,为进一步对两者发挥功效作用的物质基础及药效作用机制揭示和开发利用提供参考。

Aim To characterize the metabolic profiles of monoammonium glycyrrhizinate(MG)and diammonium glycyrrhizinate(DG)of rats in vivo by ultraperformance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UPLC-Q/TOF-MS),and the potential metabolic pathways of two components were discussed.Methods The brain tissue,plasma,urine,and feces of rats were collected at different time points after oral administration of MG and DG.Through protein precipitation,UPLC-Q/TOF-MS technology was used to conduct gradient elution with water-acetonitrile containing 0.1%formic acid as the mobile phase in the positive ion mode.The metabolites of MG and DG were predicted and characterized by mass defect filter technology combined with the mass spectrum fragmentation rules.Results MG and DG mainly existed in the form of metabolites in vivo,and a total of 20 metabolites were identified.Both of them were primarily eliminated through the intestines.A series of phase I oxidation-reduction reactions on the basic of glycyrrhetinic acid formed by the deglycosylation of the prototype compound in vivo were the main metabolic types.Glycyrrhetinic acid was the main exposed component in biological samples,which might be the key pharmacodynamic component of MG and DG in vivo.Conclusions The study clarifies the metabolic profile of MG and DG for the first time,which provides a scientific reference for further revealing the material basis and pharmacological mechanism,as well as product development.