Investigation On The Fragmentation Behaviors Of Phenolic Acids And Nitrogen Heterocvelic Compounds In Electrospray Ion Mass Spectrometry

Electrospray ionization mass spectrometry(ESI-MS)is not only an important and effective tool for structural characterization and identification,but also for investigation on the gas-phase ionic chemistry.Both protonation and deprotonation are the two very important ionization modes in ESI-MS.However,there is still short of systemic research on the fragmentation behaviors of deprotonated molecules in ESI-MS,although abundant achievements on the fragmentation mechanisms of protonated molecules have been obtained over the past few decades.In addition,fundamental research on ionization modes in ESI-MS is still not perfect.Thus,to explore the unexpected ionization reactions in ESI source and fragmentation mechanism of deprotonated molecules is very important and necessary to improve the MS characterization level and to enrich the content of MS.In this dissertation,several typical phenolic acids and nitrogen heterocyclic compounds were selected as models to investigate the side reactions occurring in ESI source and gas-phase dissociation reactions of anions in tandem MS,including the following six parts:(1)Investigation on the solvent-participating in-source aldolization reactionsUsing 2-(1,2,4-triazole-l-yl)-6-methyl-3-quinolinecarboxaldehyde as a model compound,several unusual[M+15]⁺,[M+33]⁺,and[M+47]⁺ions were observed in the positive-ion ESI source with methanol.A variety of experimental methods have showed that these unusual ions were intermediates and products of an aldolization reaction occurring in ESI source.This special reaction provides a novel approach for direct identification of drug molecules containing heteroaromatic aldehyde,and enriches a fully understanding of unexpected ionization reactions.(2)The competitive ionization modes in ESI-MS:oxidation vs protonationFormation of the unusual radical cation(M^+·)through losing an electron was obtained by ESI-MS analysis of 2-methoxy-6-(2-methyl-1H-indol-3-yl)-5H-benzo[b]carbazole in the positive-ion mode.It has been proved that formation of M^+·is due to an electrochemical oxidation reaction induced by high voltage in the spray needle.The generation of[M+H]⁺rather than M^+·is dominant in ESI-MS when a protic solvent is used;Whereas,generation of M^+·radical ion is predominent by using an aprotic ESI solvent.In addition,several ion-neutral complex-mediated reactions were obtained in the collision-induced dissociation of the corresponding[M+H]⁺ions.(3)Mechanistic study on the radical elimination of deprotonated ferulic acidTwo competitive eliminations of CO₂ and methyl radical were obtained in fragmentation of deprotonated ferulic acid.Radical loss from the even-electron ion may involve excitation of the valance electron.However,the current theoretical calculations fail to optimize the transition state and to calculate the corresponding energy barrier for these homolytic reactions,due to difficulties in treating the self-interaction of electrons.By theoretical investigations,the mechanism of methyl radical elimination has been rationalized as the electronic excitation of transient structure,which involves three steps,including elongation of O-C bond,spontaneous electronic excitation,and subsequent disruption of O-C bond.This mechanism can be used to explain formation of odd-electron ion in fragmentation of even-electron ions.(4)Investigation on competitive eliminations of methyl and benzyl radicalsBased on the results of the third part,two competitive eliminations of benzyl radical and methyl radical were investigated experimentally and theoretically in the fragmentation of deprotonated benzyl vanillate,both of which involve the electronic excitation of the transient structure.It was found that presence of an electron-withdrawing group promotes the substituted benzyl radical elimination,while that of an electron-donating group inhibits such pathway.In addition,elimination of benzyl radical was significantly affected by substitution pattern(benzyl isovanillate),and thus the two isomers can be easily differentiated solely by tandem MS.(5)Nitrobenzyl transfer reaction of deprotonated nitrobenzyl vanillatesUsing 4-nitrobenzyl vanillate as a model compound,a nitrobenzyl migration reaction,leading to elimination of CO₂ has been firstly reported in dissociation of the deprotonated molecules.Elimination of CO₂ form deprotonated nitrobenzyl vanillate involves two potential dissociation pathways:(a)electrophilic attack by benzyl;(b)radical coupling reaction mediated by triplet ion-neutral complex.DFT calculations indicated that decarboxylation of deprotonated 4-nitrobenzyl vanillate occurred predominantly via a radical route involving homolytic cleavage of the C_benzyl-O bond to give a relatively stable triplet ion-neutral complex,followed by nitrobenzyl transfer and decarboxylative coupling reaction.(6)Application of fragmentation mechanisms in the identification of unkown compounds.High-performance liquid chromatography/electrospray ionization with quadrupole time-of-flight tandem mass spectrometry(HPLC/ESI-Q-TOF-MS/MS)method was used to establish an analytical method to detect and identify phenolic constituents in the n-butanol extract of F.pandurata H.aerial roots.Thirty-six compounds,including hydroxycinnamic acid derivatives,hydroxybenzoic acid derivatives,hydroquinone glycosides,flavonoid glycosides,etc,were identified or tentatively characterized by comparing the UV spectra,accurate mass spectra,and fragmentation pathways and retrieving the reference literatures.