Determination of ion structures in structurally related compounds using precursor ion fingerprinting

Structurally-related alkaloids were analyzed by electrospray ionization/multiple stage mass spectrometry (ESI/MS ⁿ ) at varying collision energies to demonstrate a conceptual algorithm, precursor ion fingerprinting (PIF). PIF is a new approach for interpreting and library-searching ESI mass spectra predicated on the precursor ions of structurally-related compounds and their matching product ion spectra. Multiple-stage mass spectra were compiled and constructed into “spectral trees” that illustrated the compounds’ product ion spectra in their respective mass spectral stages. The precursor ions of these alkaloids were characterized and their spectral trees incorporated into an MS ⁿ library. These data will be used to construct a universal, searchable, and transferable library of MS ⁿ spectra. In addition, PIF will generate a proposed structural arrangement utilizing previously characterized ion structures, which will assist in the identification of unknown compounds.     

Studies on Triterpenoids and Flavones in Glycyrrhiza uralensis Fisch. By HPLC-ESI-MSn and FT-ICR-MSn

应用高效液相色谱质谱联用方法(HPLC-ESI-MSn)研究了甘草提取物中的七种化合物,四种三萜类化合物和三种黄酮类化合物。通过多极串联质谱(ESI-MSn)和多极串联傅里叶变换回旋共振质谱(FT-ICR-MSn)法研究了它们的碎裂规律。通过比较保留时间和质谱数据对上述七种化合物进行了归属,并阐述了其可能的质谱裂解途径。以上结果显示ESI-MSn和FT-ICR-MSn是非常有效的分析三萜类化合物和黄酮类化合物结构的工具。     

Gas‐phase fragmentation reactions of protonated benzofuran‐ and dihydrobenzofuran‐type neolignans investigated by accurate‐mass electrospray ionization tandem mass spectrometry

We have investigated gas‐phase fragmentation reactions of protonated benzofuran neolignans (BNs) and dihydrobenzofuran neolignans (DBNs) by accurate‐mass electrospray ionization tandem and multiple‐stage (MSⁿ) mass spectrometry combined with thermochemical data estimated by Computational Chemistry. Most of the protonated compounds fragment into product ions B ([M + H–MeOH]⁺), C ([ B –MeOH]⁺), D ([ C –CO]⁺), and E ([ D –CO]⁺) upon collision‐induced dissociation (CID). However, we identified a series of diagnostic ions and associated them with specific structural features. In the case of compounds displaying an acetoxy group at C‐4, product ion C produces diagnostic ions K ([ C –C₂H₂O]⁺), L ([ K –CO]⁺), and P ([ L –CO]⁺). Formation of product ions H ([ D –H₂O]⁺) and M ([ H –CO]⁺) is associated with the hydroxyl group at C‐3 and C‐3′, whereas product ions N ([ D –MeOH]⁺) and O ([ N –MeOH]⁺) indicate a methoxyl group at the same positions. Finally, product ions F ([ A –C₂H₂O]⁺), Q ([ A –C₃H₆O₂]⁺), I ([ A –C₆H₆O]⁺), and J ([ I –MeOH]⁺) for DBNs and product ion G ([ B –C₂H₂O]⁺) for BNs diagnose a saturated bond between C‐7′ and C‐8′. We used these structure‐fragmentation relationships in combination with deuterium exchange experiments, MSⁿ data, and Computational Chemistry to elucidate the gas‐phase fragmentation pathways of these compounds. These results could help to elucidate DBN and BN metabolites in in vivo and in vitro studies on the basis of electrospray ionization ESI‐CID‐MS/MS data only.     

Effect of pH on the Metabolism of Aconitine under Rat Intestinal Bacteria and Analysis of Metabolites Using HPLC/MS-MSn Technique

A semi‐quantitative method of mass spectrometry (MS) has been described for the analysis of metabolites of aconitine by rat intestinal bacteria at different pH. At pH 7.0, the rat intestinal bacteria exhibit optimal activity for the metabolism of aconitine. A high‐performance liquid chromatography‐electrospray ionization multiple‐stage mass spectrometry (HPLC/ESI‐MSⁿ) method has been applied to investigate the characteristic product ions of metabolites. Then, the logical fragmentation pathways of metabolites have been proposed. By comparing the retention time (t_R) of HPLC and the ESI‐MSⁿ data with the data of standard compounds and reports from literature, ten metabolites have been identified and a distinctive metabolite (15‐deoxyaconitine) has been deduced first time. The experimental results demonstrate that HPLC/ESI‐MSⁿ is a specific and useful method for the identification of metabolites of aconitine. Also, in the present paper, the HPLC‐MS method was introduced to determine the synthetical metabolite prior to the study of the toxicity by the method of Bliss.     

二肽衍生物的电喷雾质谱研究

基于HIV整合酶核心结构域,合成了以HIV整合酶为靶标的二肽衍生物,采用多级质谱技术（二级、三级）研究二肽衍生物在质谱条件下的化学键断裂途径,发现主要的断裂方式为：氨基与羰基间的NH-CO键的断裂以及N-(苯并噻唑-2-基)甲酰氨基与亚甲基间的CO-C间的断裂。     

Alkaloids from the Roots of Stemona cochinchinensis

Phytochemical investigation of the roots of Stemona cochinchinensis led to the isolation and structure elucidation of a new pyrido[1,2‐a]azepine‐type alkaloid, stemocochinamine ( 1 ), and of four new pyrrolo[1,2‐a]azepine‐type alkaloids, bisdehydrostemocochinine ( 2 ), isobisdehydrostemocochinine ( 3 ), neostemocochinine ( 4 ), and isoneostemocochinine ( 5 ), together with six known alkaloids. Their structures were established on the basis of extensive 1D‐ and 2D‐NMR analyses in combination with HR‐MS experiments.     

Bisbenzopyrans and alkaloids from the roots of Stemona cochinchinensis

Two new bisbenzopyrans, namely 3,3'-bis(3,4-dihydro-4-hydroxy-6,8-dimethoxy-2H-1-benzopyran) (1), and 8'-methoxy-3,3'-bis(3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran) (2), together with known 3,3'-bis(3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran) (3), as well as three known pyrido[1,2-alpha]azapine alkaloids, stemokerrin (4), oxystemokerrin (5) and oxystemokerrin-N-oxide (6), were isolated and identified from the roots of Stemona cochinchinensis. The structures of these compounds were elucidated by 1D- and 2D-NMR spectra and other spectroscopic studies.     

Dereplication of known pregnane glycosides and structural characterization of novel pregnanes in Marsdenia tenacissima by high‐performance liquid chromatography and electrospray ionization‐tandem mass spectrometry

In the search for novel natural products in plants, particularly those with potential bioactivity, it is important to efficiently distinguish novel compounds from previously isolated, known compounds, a process known as dereplication. In this study, electrospray ionization‐multiple stage tandem mass spectrometry (ESI‐MSⁿ) was used to study the behaviour of 12 pregnane glycosides and genins previously isolated from Marsdenia tenacissima, a traditional Chinese medicinal plant, as a basis for dereplication of compounds in a plant extract. In addition to [M + Na]⁺ and [M + NH₄]⁺ ions, a characteristic [M‐glycosyl + H]⁺ ion was observed in full‐scan mode with in‐source fragmentation. Sequential in‐trap collision‐induced dissociation of [M + Na]⁺ ions from 11,12‐diesters revealed consistent preferred losses of substituents first from C‐12, then from C‐11, followed by losses of monosaccharide fragments from the C‐3 tri‐ and tetrasaccharide substituents. A crude methanol extract of M. tenacissima stems was analysed using high‐performance liquid chromatography coupled to ESI‐MS. Several previously isolated pregnane glycosides were dereplicated, and the presence of an additional nine novel pregnane glycosides is predicted on the basis of the primary and fragment ions observed, including two with a previously unreported C₄H₇O C‐11/C‐12 substituent of pregnane glycosides. This study is the first report of prediction of the structures of novel pregnane glycosides in a crude plant extract by a combination of in‐source fragmentation and in‐trap collision‐induced dissociation and supports the usefulness of LC‐ESI‐MSⁿ not only for dereplication of active compounds in extracts of medicinal plants but also for detecting the presence of novel related compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Study of active naphtodianthrone St John's Wort compounds by electrospray ionization Fourier transform ion cyclotron resonance and multi‐stage mass spectrometry in sustained off‐resonance irradiation collision‐induced dissociation and infrared multiphoton dissociation modes

Five well‐known active naphtodianthrone constituents of Hypericum perforatum (St John's Wort) extracts have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI‐FTICRMS) and ESI‐FTICRMSⁿ. The studied compounds were hypericin, pseudohypericin, protohypericin, protopseudohypericin (biosynthetic precursors of the two former compounds, respectively) and isopseudohypericin (alkaline degradation product of pseudohypericin). Dissociation mass spectrometry measurements performed on the [M–H]^? ion presented a variable efficiency as a function of the used activation mode. Sustained off‐resonance irradiation collision‐induced dissociation (SORI–CID) only led to a restricted number of fragment ions. In contrast, IRMPD ensured the detection of numerous product ions. Ions detected in ESI‐FTICRMS and ESI‐FTICRMSⁿ experiments were measured with a very high mass accuracy (typically mass error is lower than 0.5 mDa at m/z close to 500) that allowed unambiguous formulae to be assigned to each signal observed in a mass spectrum. In spite of similar structures, specific fragmentation patterns were observed for the different compounds investigated. This study may be useful in the future to characterize in natural extracts these compounds (or derivatives of these compounds) by liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments by considering the MS/MS transitions highlighted in this paper. Copyright © 2009 John Wiley & Sons, Ltd.     

Mass spectrometric profiling of valepotriates possessing various acyloxy groups from Valeriana jatamansi

Valepotriates, plant secondary metabolites of the family Valerianaceae, contain various acyloxy group linkages to the valepotriate nucleus and exhibit significant biological activities. Identification of valepotriates is important to uncover potential lead compounds for the development of new sedative and antitumor drugs. However, making their structure elucidation by nuclear magnetic resonance (NMR) experiments is too difficult to be realized because of the overlapped carbonyl carbon signals of acyloxy groups substituted at different positions. Thus, the mass spectrometric profiling of these compounds in positive ion mode was developed to unveil the exact linkage of acyloxy group and the core of valepotriate. In this study, electrospray ionization tandem multistage mass spectrometry (ESI‐MS/MSⁿ) in ion trap and collision‐induced dissociation tandem MS were used to investigate the fragmentation pathways of four types of valepotriates in Valeriana jatamansi, including 5‐hydroxy‐5,6‐dihydrovaltrate hydrin (5‐hydroxy‐5,6‐dihydrovaltrate chlorohydrin), 5,6‐dihydrovaltrate hydrin (5,6‐dihydrovaltrate chlorohydrin), 5‐hydroxy‐5,6‐dihydrovaltrate and valtrate hydrin (valtrate chlorohydrin). The high‐resolution mass spectrum (HRMS) data of all the investigated valepotriates from quadrupole time‐of‐flight MS/MS were used as a supportive of the fragmentation rules we hypothesized from ion‐trap stepwise MSⁿ. As a result, the loss sequence of acyloxy groups and the abundance of key product ions, in combination with the characteristic product ions corresponding to the valepotriate nucleus, could readily differentiate the four different types of valepotriates. The summarized fragmentation rules were also successfully exploited for the structural characterization of three new trace valepotriates from V. jatamansi. The results indicated that the developed analytical method could be employed as a rapid, effective technique for structural characterization of valepotriates, especially for the trace compounds that could not be identified by NMR techniques. This study may also arouse interest for further structural analysis of other valepotriate‐containing type herbal medicines. Copyright © 2015 John Wiley & Sons, Ltd.     

MassBank: a public repository for sharing mass spectral data for life sciences

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron‐ionization mass spectrometry(EI‐MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)‐MSⁿ data of 2337 authentic compounds of metabolites, 11 545 EI‐MS and 834 other‐MS data of 10 286 volatile natural and synthetic compounds, and 3045 ESI‐MS² data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI‐MS² data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass‐to‐charge ratio are optimized to the ESI‐MS² data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI‐MS² data on an identical compound under different collision‐induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21–23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data. Copyright © 2010 John Wiley & Sons, Ltd.     

Quality assessment of Cortex Phellodendri by high‐performance liquid chromatography coupled with electrospray ionization mass spectrometry

A simple method based on liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (LC‐DAD‐ESI‐MS) was developed for the quality assessment of Cortex Phellodendri (CP), which was mainly derived from two species of Phellodendron chinense Schneid and Phellodendron amurense Rupr. Total 41 compounds, including 14 phenols, 24 alkaloids and three liminoidal triterpenes were identified or tentatively characterized from the 75% methanol extract of CP samples by online ESI‐MSⁿ fragmentation and UV spectra analysis. Among them, two phenols and six alkaloids were simultaneously quantified using HPLC‐DAD method. The validated HPLC‐DAD method showed a good linearity, precision, repeatability and accuracy for the quantification of eight marker compounds. Furthermore, the plausible fragmentation pathway of the representative compounds were proposed in the present study. The differences of the chemical constituents content and the comprehensive HPLC profiles between the two CP species using LC‐DAD‐ESI‐MS method are reported for the first time, indicating that the CP drugs from different resources should be used separately in the clinic. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization and identification of iridoid glucosides,flavonoids and anthraquinones in Hedyotis diffusa by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

The multiple bioactive constituents in Hedyotis diffusa Willd. (H. diffusa) were extracted and characterized by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MSⁿ). The optimized separation condition was obtained using an Agilent ZorBax SB‐C₁₈ column (4.6×150 mm, 5 μm) and gradient elution with water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid), under which baseline separation for the majority of compounds was achieved. Among the compounds detected, 14 iridoid glucosides, 10 flavonoids, 7 anthraquinones, 1 coumarin and 1 triterpene were unambiguously identified or tentatively characterized based on their retention times and mass spectra in comparison with the data from standards or references. The fragmentation behavior for different types of constituents was also investigated, which could contribute to the elucidation of these constituents in H. diffusa. The present study reveals that even more iridoid glycosides were found in H. diffusa than hitherto assumed. The occurrence of two iridoid glucosides and five flavonoids in particular has not yet been described. This paper marks the first report on the structural characterization of chemical compounds in H. diffusa by a developed HPLC‐ESI‐MSⁿ method.     

Tandem mass spectrometry based investigation of cinnamylideneacetophenone derivatives: valuable tool for the differentiation of positional isomers

Cinnamylideneacetophenones have been extensively used as versatile starting materials in numerous different transformations. The structural characterization of this type of compounds is, therefore, of crucial importance since it can give information on the chemistry, reactivity and also the potential biological activity of this type of compounds. Thus, 24 derivatives were systematically studied by tandem mass spectrometry (MS²) with electrospray ionization (ESI), in positive ion mode. The protonated molecules, [M + H]⁺, formed under ESI conditions were induced to dissociate and the fragmentation patterns were studied. The information collected provided important structural information on the type of substituents present and constitute an important database concerning this family of compounds. Overall, it was found that the substitution pattern of the cinnamylideneacetophenone derivatives changes the ESI‐MS² fragmentation considerably. These results indicate that ESI‐MS² is a useful technique for distinguishing positional isomers of these cinnamylideneacetophenone derivatives. Copyright © 2011 John Wiley & Sons, Ltd.     

Fragmentation of deprotonated cyclic dipeptides by electrospray ionization mass spectrometry

The fragmentation pathways of deprotonated cyclic dipeptides have been studied by electrospray ionization multi‐stage mass spectrometry (ESI‐MSⁿ) in negative mode. The results showed that the fragmentation pathways of deprotonated cyclic dipeptides depended significantly on the different substituents, the side chains of amino acid residues at the diketopiperazine ring. In the spectra of deprotonated cyclic dipeptides, the ion [M? H? substituent radical]^? was firstly observed in the ESI mode. The characteristic fragment ions [M? H? substituent radical]^? and [M? H? (substituent? H)]^? could be used as the symbols of particular cyclic dipeptides. The hydrogen/deuterium (H/D) exchange experiment, the high‐resolution mass spectrometry (Q‐TOF) and theoretical calculations were used to rationalize the proposed fragmentation pathways and to verify the differences between the fragmentation pathways. The relative Gibbs free energies (ΔG) of the product ions and possible fragmentation pathways were estimated using the B3LYP/6–31++G(d, p) model. The results have some potential applications in the structural elucidation and interpretation of the mass spectra of homologous compounds and will enrich the gas‐phase ESI‐MS ion chemistry of cyclic dipeptides. Copyright © 2009 John Wiley & Sons, Ltd.     

两类三组份反应产物：1,3-恶嗪及嘧啶-2-酮类化合物的质谱学行为研究

电喷雾质谱被应用于分辨2-氨基-1,3-恶嗪及六氢化-4-苯基-吡喃[2,3-d]嘧啶-2-酮的杂环结构。两类化合物均为三组份反应的产物,且其杂环的结构很难用NMR判断。实验首次系统研究了两类化合物的质谱学行为（包括氘代实验和高分辨质谱研究）,发现前者在CID实验中丢失CH2N2和HCNO,而后者为直接丢失尿素。这些特征丢失为该类衍生物的结构判断,尤其是高通量的合成产物分析提供了重要的依据。     

Identification of new trace triterpenoid saponins from the roots of Panax notoginseng by high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Triterpenoid saponins are the major bioactive constituents of Panax notoginseng. In the study reported here, the fragmentation behavior of triterpenoid saponins from P. notoginseng was investigated by electrospray ionization tandem mass spectrometry (ESI‐MSⁿ)and high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC/ESI‐MSⁿ). Analyses revealed that product ions from glycosidic and cross‐ring cleavages can give a wealth of structural information regarding the nature of the aglycone, sugar types, the sequence and linkage information of sugar units. It is noted that different glycosylation positions remarkably influenced the fragmentation behaviors, which could assist in the differentiation of saponin analogues. To rationalize this characteristic, the collision energy required for various glycosidic cleavages was investigated. According to the summarized fragmentation rules, identification of triterpenoid saponins from the roots of P. notoginseng could be fulfilled, even when reference standards were unavailable. Furthermore, minor and trace constituents were enriched and detected by eliminating the major constituents in one of the saponin fractions. As a result, a total of 151 saponins, including 56 new trace ones, were identified or tentatively characterized from saponin fractions based on their retention times, HPLC/HRMS, HPLC/ESI‐MSⁿ fragmentation behaviors and comparison with literature data. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives using electrospray ionization multi‐stage mass spectrometry

N‐Boc/Fmoc/Z‐N′‐formyl‐gem‐diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro‐inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion‐trap multi‐stage mass spectrometry (MSⁿ). The MS² collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N‐Fmoc/Z‐amino acids, dipeptide and tripeptide acids show the [M + Na‐NH₂CHO]⁺ ion, arising from the loss of formamide, as the base peak. Differently, the MS² CID spectra of [M + Na]⁺ ion of all the N‐Boc derivatives yield the abundant [M + Na‐C₄H₈]⁺ and [M + Na‐Boc + H]⁺ ions because of the loss of isobutylene and CO₂ from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N‐protected dipeptidyl and tripeptidyl‐N′‐formamides is provided by MS² and subsequent MSⁿ experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M‐H]^?, [M + HCOO]^? and [M + Cl]^? ions, the presence of in‐source CID fragment ions deriving from the involvement of the N‐protecting group. Furthermore, MSⁿ spectra of [M + Cl]^? ion of N‐protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C‐terminal gem‐diamino residue. The present paper represents an initial attempt to study the ESI‐MS behavior of these important intermediates and lays the groundwork for structural‐based studies on more complex partially modified retro‐inverso peptides. Copyright © 2013 John Wiley & Sons, Ltd.     

Microsynthesis and mass spectral study of Chemical Weapons Convention related 2‐alkyl‐1,3,6,2‐dioxathiaphosphocane‐2‐oxides

Chemical Weapons Convention (CWC)‐related compounds where the phosphorus atom is part of a ring have very limited representation in mass spectral libraries and the open literature. Here we report electron ionization (EI), chemical ionization (CI) and electrospray ionization tandem mass spectrometry (ESI‐MSⁿ) spectra and retention indices for 2‐alkyl‐1,3,6,2‐dioxathiaphosphocane‐2‐oxides (alkyl C₁ to C₃) which are new cyclic chemicals covered under the CWC. The EI mass spectra show a pattern of ion fragmentation that is similar to that of other cyclic phosphonates in that loss of the alkylphosphonic acid as a neutral loss is more important than the presence of the protonated alkylphosphonic acid. In contrast to other cyclic phosphonates, the 2‐alkyl‐1,3,6,2‐dioxathiaphosphocane‐2‐oxides show almost no protonated alkylphosphonic acid and as a result the spectra do not carry the same distinctive signature of the phosphorus–carbon bond that is required for the chemical to be covered under the CWC. Copyright © 2008 John Wiley & Sons, Ltd.     

Two New‐Skeleton Compounds from Sarcandra glabra

Two new‐skeleton compounds, sarcaboside A ( 1 ) together with its artifact sarcaboside B ( 2 ), were isolated from the whole plant of Sarcandra glabra. Their structures were elucidated on the basis of extensive spectroscopic analysis as well as HR‐ESI‐MSⁿ study.