A Methodological Study On The Identification Of Chemical Components In Herbs By LC/MS Technologies

The Chinese herbal medicine is a treasury of natural products,and fascinating more and more researchers to explore the ancient secret in it.The development of LC/MS technology may be a key to this treasury after it's mature in late 90s of last century.In this work,three herbs which have shown solid curative effects to cardiovascular diseases,cancers and inflammations were selected as our research subjects and analyzed by many LC/MS technologies.Our results will be a good example of how natural compounds are identified by ESI-MSⁿ.In order to improve the speed,reduce human labor,and make it more intelligent in handling LC/MS data,new algorithms and programs were coded and a database of multi-stage mass spectra also established even though the current version is very primary.The main contents and our contributions are summarized as following:(1) A LC/DAD/MSⁿ method was developed to analyze the phenolic compounds of Erigeron breviscapus and Smallanthus sonchifolius.A total of 53 compounds were identified or tentatively characterized based on their UV and mass spectra.These compounds included caffeoylquinic acids(CQAs),CQA glucosides,malonyl-CQAs,acetyl-CQAs,caffeoyl-2,7-anhydro-3-deoxy-2-octulopyranosonic acids(CDOAs),caffeoyl-2,7-anhydro-2-octulopyranosonic acids(COAs), flavones,flavonols and flavonones.Most of them were reported for the first time from E. breviscapus and nineteen of them belonged to new compounds.The MSⁿ spectra of CQA glucosides were similar to CQAs and they were discriminated by their retention times.Fragment ions(^2'5O^-,^3,6O^- and ^4,6O^-) corresponding to ring cleavage were shown in MSⁿ spectra of CDOAs and COAs,characteristic of this class of compounds.(2) Eighteen isoprenylated flavonoids(8 flavanones,3 flavanols,and 7 chalcones) isolated from Kushen or synthesized were studied by positive and negative ion electrospray ionization multi-stage tandem mass spectrometry(ESI-MSⁿ).Plausible fragmentation patterns were obtained by comparing their MSⁿ spectra with each other,which were further supported by high-resolution MS data and two model compounds.It was shown that the 2'-OH group would make the C-ring of flavonoids studied more labile through a six-membered mechanism,resulting in base peaks of ^1,3A+(positive mode) and ^1,4A-(negative mode).In addition,the 2'-OH is also responsible for the neutral loss of water in(+)ESI/MS² of flavanones.The neutral loss of water(or methanol) in (-)ESI/MS² of flavanols was elucidated by a E2 elimination mechanism.Different relative abundances of ^1,3A⁺ and S⁺ in(+)ESFMS² spectra were used to discriminate flavanones with their open-ring products,chalcones,since the equilibrium for flavanone??chalcone isomerization in ESI ion source could not be obtained in positive mode.(3) The main components(isoquinoline alkaloids,diterpenoids and steroids) in crude extracts of Chinese herb Jin-Guo-Lan,prepared from the roots of Tinospora sagittata and Tinospora capillipes,were explored by liquid chromatography/electrospray ionization multistage mass spectrometry coupled with diode array detection(LC-DAD/ESI-MSⁿ).In positive ion mode,the three types of compounds showed very different characteristic ions:strong[M]⁺ or[M+H]⁺ ions were observed for isoquinoline alkaloids;[M+NH₄]⁺ and/or[M-CO₂]⁺ for diterpenoids; [M+H-nH₂O]⁺(n=1-3) for steroids.These adduct ions and/or fragments were used to deduce the mass and categories of known and unknown components in crude extracts,and their structures were further confirmed by ESI-MSⁿ in positive ion mode.Moreover,UV absorption peaks obtained from diode array detection provided useful functional group information to aid the MSⁿ-based identification.As a result,11 compounds were unambiguously identified by comparing with standard compounds and 13 compounds were tentatively identified or deduced according to their MSⁿ data.Two of these compounds(13-hydroxycolumbamine and 13-hydroxyjatrorrhizine) were found to be new compounds and another one (13-hydroxypalmatine) was firstly detected as natural products.Although the roots of Tinospora sagittata and Tinospora capillipes have almost identical compounds,the content of the compounds in them is dramatically different,suggesting the necessity of further comparison the bioactivities of the two species.(4) A software "raw2txt" was programmed to extract full scan mass spectrum data from binary raw files of Finnigan.This is a basic tool for users to develop other algorithms about LC/MS data.The frequency-based mass selection algorithm(FMSA) can beat any other reported methods in discrimination of noise and signals.The programs "AddCal" and "MS2table" are helpful for users to determine the construction of adduct ions and format the MSⁿ data for publication.(5) A novel quality control method was developed based on the two-way data from LC/MS, in which both quantitative and qualitative information was included.(6) A multi-stage mass spectra database for natural products(MMSDNP) was constructed.It can be used as a laboratory MS information management system or assist users to characterize unknown compounds quickly.