摘要
本论文主要开展了苯乙醇苷等类天然产物的质谱分析方法及其混合物体系的LC-MS与NMR相关谱分析方法研究,实现了复杂混合物不完全分离的同步结构鉴定。首先,在负离子模式下对氢醌苷和苯乙醇苷类化合物进行了系统的ESI-MS~n谱分析研究,掌握了这两类化合物结构特征与质谱裂解行为之间的相关性,归纳及总结它们的ESI-MS~n裂解特征,建立了简便、快速鉴别氢醌苷和苯乙醇苷类化合物的质谱分析方法。在前期建立的LC-MS与~1H NMR的平行动态谱(NMR/LC-MS PDS)和正交相关谱(NMR/LC-MS CHS)方法的基础上,成功地将这种新型谱学分析方法应用于小蜡树及连翘提取物,拓展了NMR/LC-MS PDS与NMR/LC-MS CHS谱学分析方法的应用范围;同时结合HPLC-MS~n,2D NMR谱等方法,实现了复杂混合物体系组分群无需完全分离,高效、多容量的结构鉴定。运用上述方法共分析鉴定两份提取物中19个成分的结构,其中连翘提取物中有3个成分为新化合物,小蜡树提取中有4个成分为该属植物内首次发现。
本论文的研究内容主要包括以下四个部分:
1.氢醌苷类化合物的质谱裂解规律研究
采用(-)ESI-MS~n方法,系统地研究了一系列氢醌苷类化合物的质谱裂解规律,发现其结构特征与质谱行为的相关性。其中氢醌单糖苷及氢醌1,4-二-O-糖苷主要发生糖苷键的均裂与异裂反应,产生自由基苷元离子([Y_0-H]~-)与苷元离子(Y_0~-)。研究还发现苯环上甲氧基的取代数目及其位置对[Y_0-H]~-/Y_0~-离子强度比值产生显著的影响。此外,通过细致的考察及优化碰撞能量,获得[Y_0-H]~-与Y_0~-离子相对强度的特异性差异,成功用于糖基化位置不同的氢醌单糖苷同分异构体的结构鉴别。此外,氢醌O-双糖苷类化合物主要发生糖链部分的交叉环切除反应,产生与糖链结构相关的系列特征离子。通过氢醌苷类化合物裂解规律的研究,建立了快速分析这类化合物的质谱分析方法,并为混合物中同类成分的结构鉴定提供了可靠的分析依据。
2.苯乙醇苷类化合物的质谱裂解规律研究
采用(-)ESI-MS~n方法,对不同类型的苯乙醇苷类化合物的裂解行为进行了细致地研究,其中包括苯乙醇单糖苷、芹糖苯乙醇苷及咖啡酰基苯乙醇苷等类化合物,探讨了各类化合物结构特征与质谱行为的相关性及其变化规律。研究发现苯乙醇单糖苷类化合物由于其糖基化的位置及苯环上C-3位取代基的不同而表现出不同的裂解行为;咖啡酰基苯乙醇苷类化合物,则产生一系列与咖啡酰基有关的特征离子。通过ESI-MS~n谱分析能够有效地鉴定各种类型的苯乙醇苷类化合物,并为指导混合物中同类成分的结构鉴定提供了有力的分析依据。
3.小蜡树提取物组分群的NMR与LC-MS平行动态谱和正交相关谱分析方法研究
将小蜡树乙醇提取物作为研究对象,首次以凝胶柱色谱为主要分离手段,获取含量呈连续动态变化的系列混合物。对于系列流份分别进行LC-MS~n和~1H NMR谱测定,并采用MATLAB软件编写的数据处理程序,对原始数据进行处理与分析,构成NMR/LC-MS PDS和NMR/LC-MS CHS谱。通过综合分析,获取多组分混合物中同一成分的质荷比(m/z)和化学位移值(δ)等相关性信息,利用这些信息并结合HPLC-MS~n、~1H-~1H COSY、1D或2D NOESY谱等分析方法对这些系列流份中的氢醌苷类及苯乙醇苷类成分进行了结构分析研究。实现了小蜡树提取物中12个成分不完全分离的同步结构鉴定,其中包括3个氢醌苷类化合物,8个苯乙醇苷类化合物,以及一个香草酸苷类化合物,其中有4个成分为首次从本属植物中发现。
4.连翘提取物组分群的NMR与LC-MS平行动态谱和正交相关谱分析方法研究
以常用中药连翘水提取物为研究对象,利用制备型高效液相色谱技术获取含量呈连续动态变化的系列混合物,并分别进行LC-MS~n和~1H NMR谱测定。然后采用MATLAB软件编写的数据处理程序,构成NMR/LC-MS PDS和NMR/LC-MS CHS谱,并结合HPLC-MS~n及~1H-~1H COSY,1D-TOCSY和2D NOESY等谱学方法对这些系列混合物中苯乙醇苷类成分进行结构分析研究,实现了混合物中不同组分的同步结构鉴定。从提取物LQ-7中成功鉴定了出7个成分,包括6个苯乙醇苷类化合物,一个黄酮苷类化合物;其中有3个成分为新化合物,有一成分为首次发现的苯乙醇苷二聚体生物碱。
For the purpose of simultaneous structural identification of natural products in crude extracts,the fragmentation behavior of phenylethanoid glycosides etc.and correlation spectroscopy of LC-MS/NMR of their complex mixtures were studied.First of all,the hydroquinone glycosides and phenylethanoid glycosides were studied systematically by electrospray ionization tandem mass spectrometry in negative ion mode in order to finding out the relationship between their structural features and the corresponding characteristic fragmentation behavior.The fragmentation rules of these known compounds were summarized.Consequently,the NMR/LC-MS PDS and NMR/LC-MS CHS of the crude extracts of Fraxinus sieboldiana and Forsythia Suspensa were established.Combined with HPLC-MS~n and 2D NMR spectra,total nineteen constituents in these two extracts were identified successfully,in which three compounds were new from Forsythia Suspensa extract and four compounds were found from the genus Fraxinus for the first time.
1.Characteristic fragmentation behavior of hydroquinone glycosides using electrospray ionization tandem mass spectrometry
The characteristic fragmentation behavior of hydroquinone glycosides from Fraxinus sieboldiana was investigated systematically by electrospray ionization tandem mass spectrometry in negative ion mode.The characteristic fragmentation reaction of these compounds was through the homolytic and heterolytic cleavage of the O-glycosidic bond to produce radical aglycone ion([Y_0-H]~(-·)) and aglycone ion(Y_0~-),respectively.It was found that the relative abundance of the[Y_0-H]~(-·) ion and Y_0~- ion of these compounds exhibited a clear difference depending on the glycosylation position,number and position of the methoxy group.In addition,unambiguous differentiation between the mono-O-glycosides isomers which differ in glycosylation position was achieved by comparing the relative abundance of[Y_0-H]~(-·) and Y_0~- ions with the optimized collision energy.For hydroquinone O-diglycosides,the fragmentation behavior was characterized by cross-ring cleavage of the sugar moieties,producing a series of ions related to the sugar moieties.The results of this study can facilitate the rapid determination of hydroquinone glycosides in crude plant extracts.
2.Characteristic fragmentation behavior of phenylethanoifl glycosides using electrospray ionization tandem mass spectrometry
The characteristic fragmentation behavior of various type phenylethanoid glycosides involving glucosyl groups,apiofuranosyl group and caffeyl group substituents was investigated systematically by electrospray ionization tandem mass spectrometry in negative ion mode.The relationship between their structural features and the corresponding characteristic fragmentation behavior was investigated.It was found that the glycosylation position and substituent group at C-3 significantly influenced the fragmentation behavior of the phenylethanoid mono-O-glycosides.For phenylethanoid glycosides with caffeyl group,there were series of ions related to caffeyl group in their MS/MS spectra.The various type phenylethanoid glycosides could be identified by investigation of their ESI-MS~n spectra.And the results of this study can be useful for the rapid determination of phenylethanoid glycosides in crude plant extracts.
3.NMR/LC-MS PDS and NMR/LC-MS CHS of extract of Fraxinus sieboldiana
Based on the incomplete separation analysis strategy,the extract of Fraxinus sieboldiana was separated into series of fractions with the concentration dynamic variation of constituents by gel chromatography.The LC-MS and ~1H NMR data of series of fractions were acquired and then processed by MATLAB to form NMR/LC-MS PDS and NMR/LC-MS CHS.The correlation relationship between ions(m/z) and chemical shift(δ) deriving from the same molecule can be extracted simultaneously from the mixture spectra.Combined with HPLC-MS~n,~1H-~1H COSY,1D or 2D NOESY spectra, twelve constituents including three hydroquinone glycosides,eight phenylethanoid glycosides and one vanillic glycoside were identified successfully,in which four compounds were found from genus Fraxinus for the first time.
4.NMR/LC-MS PDS and NMR/LC-MS CHS of extract of Forsythia Suspensa
The extract of Forsythia Suspensa was separated into series of fractions with the concentration dynamic variation of constituents by RP preparative chromatography.The LC-MS and ~1H NMR data of series of fractions were processed by MATLAB to form NMR/LC-MS PDS and NMR/LC-MS CHS,the correlation relationship between MS and ~1H NMR data deriving from the same molecule can be extracted from the mixture spectra. Combined with HPLC-MS~n,~1H-~1H COSY,2D NMR spectra,seven constituents including six phenylethanoid glycosides and one flavonol were identified successfully,in which three compounds were new.
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