基于HPLC-HRMS/MS~n/QqQ技术的人参皂苷Rb_1化学转化产物的结构与途径分析

利用液相色谱-质谱联用技术分析了Keggin型12-磷钨酸化学转化人参皂苷Rb1产物的结构与转化途径.基于高效液相色谱对转化产物的快速分离,利用Q Exactive高分辨质谱的Full MS-AIF模式快速鉴定了产物结构,并利用多级串联质谱进行结构验证.进一步结合人参皂苷异构体在反向C18色谱柱上的相对保留时间,快速分析鉴定出Rb1的10种转化产物为20(S)-Rg3,20(R)-Rg3,20(S)-25-OH-Rg3,20(R)-25-OH-Rg3,25-OH-Rk1,25-OH-Rg5,Rg5,Rk1,(20S,25)-环氧-Rg3和(20R,25)-环氧-Rg3.根据转化产物的结构初步推断了人参皂苷的转化途径:在12-磷钨酸产生的酸性环境中,Rb1主要通过C20位去糖基化、差向异构化和烯烃链的水合、消除及环合反应转化为稀有皂苷.采用三重四极杆质谱的选择反应监测模式准确定量分析了Rb1的转化效率和稀有皂苷20(S)-Rg3,20(R)-Rg3,Rk1和Rg5的产率.定量分析结果显示,与生物转化相比,12-磷钨酸对Rb1有更高的转化效率,反应40 min后转化率达到100%.本文结果表明,HPLC-HRMS/MSn/Qq Q技术是人参皂苷等天然产物结构解析与定量分析的有效方法.

Structure and Pathway Research on Chemical Transformation of Ginsenoside Rb1 via HPLC-HRMS/MSn/QqQ Technique

Keggin-type dodeca tungstophosphoric acid(HPW) was used in the chemical transformation of ginsenoside Rb1 . The aim of this research is to analyze the structure of the transformed products and transformation pathways based on high-performance liquid chromatography coupled with three kinds of mass spectrometry( HPLC-MS) , the potential of which in qualitative and quantitative analysis of natural product was also developed. Q Exactive, a kind of high-resolution MS( HRMS) , was used for the rapid structural identifi-cation of transformed products. Multistage tandem MS ( MSn ) was further applied to verifying the identified structure. Ten transformed products, 20 ( S )-Rg3 , 20 ( R )-Rg3 , 20 ( S )-25-OH-Rg3 , 20 ( R )-25-OH-Rg3 , 25-OH-Rk1, 25-OH-Rg5, Rg5, Rk1, (20S,25)-epoxy-Rg3 and(20R,25)-epoxy-Rg3 were rapidly identified in all. In HPW-generated acidic solution, ginsenoside Rb1 was chemically transformed through deglycosyla-tion, epimerization at C20 as well as hydration, elimination and cyclization reactions on the aglycone. Triple quadrupole(QqQ) MS was further used in its SRM mode for the accurately quantitative analysis of the conver-sion of Rb1 and the yield of 20(S)-Rg3, 20(R)-Rg3, Rk1 and Rg5. Rb1 was completely transformed to rare ginsenosides within 40 min. Based on the quantitative analysis results, HPW exhibited a much higher activity towards the transformation of Rb1 in comparison with biotransformation, and it is a kind of efficient reagent for the chemical transformation of ginsenoside Rb1. What's more, HPLC-HRMS/MSn/QqQ technique was proved to be effective for the qualitative and quantitative analysis of the natural products such as ginsenosides.