Structural characterization of constituents with molecular diversity in fractions from Lysidice brevicalyx by liquid chromatography/diode-array detection/electrospray ionization tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance

A combination of electrospray ionization tandem mass spectrometry with high-performance liquid chromatography (HPLC/ESI-MSn), and hyphenation of liquid chromatography to nuclear magnetic resonance spectroscopy (HPLC/NMR), have been extensively utilized for on-line analysis of natural products, analyzing metabolite and drug impurity. In our last paper, we reported an on-line analytical method for structural identification of trace alkaloids in the same class. However, the structural types of the constituents in plants were various, such as flavanoids, terpenoids and steroids. It is important to establish an effective analytical method for on-line structural identification of constituents with molecular diversity in extracts of plants. So, in the present study, the fragmentation patterns of some isolated stilbenes, phloroglucinols and flavanoids from Lysidice rhodostegia were investigated by ESI-MSn. Their fragmentation rules and UV characteristics are summarized, and the relationship between the spectral characteristics, rules and the structures is described. According to the fragmentation rules, NMR and UV spectral characteristics, 24 constituents of different types in the fractions from L. brevicalyx of the same genus were structurally characterized on the basis of HPLC/HRMS, HPLC-UV/ESI-MSn, HPLC/1H NMR and HPLC/1H-1H COSY rapidly. Of these, six (10, 13, 14, 16, 17 and 23) are new compounds and all of them are reported from L. brevicalyx for the first time. The aim is to develop an effective analytical method for on-line structural identification of natural products with molecular diversity in plants, and to guide the rapid and direct isolation of novel compounds by chemical screening.     

Rapid separation and identification of phenolic and diterpenoid constituents from Radix Salvia miltiorrhizae by high-performance liquid chromatography diode-array detection, electrospray ionization time-of-flight mass spectrometry and electrospray ionization quadrupole ion trap mass spectrometry

High-performance liquid chromatography-diode array detection (HPLC-DAD), electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) and electrospray ionization quadrupole ion trap mass spectrometry (HPLC-ESI-MSn) were used for the isolation, identification and structural analysis of water-soluble phenolic and nonpolar diterpenoid constituents in Dan-shen (Radix Salvia miltiorrhizae) which was prepared by sonication in 70% methanol. Mass spectra were obtained by ESI-TOF-MS and electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT-MS). A formula database of known constituents in Dan-shen was established and most constituents were rapidly identified by HPLC-DAD/ESI-TOF-MS by matching their accurate molecular masses with the formulae of the compounds in the database. Compounds with the same molecular formula could not be differentiated by TOF-MS; however, QIT-MS could differentiate those compounds and elucidate their structures based on their characteristic fragmentation. HPLC-DAD, HPLC/ESI-TOF-MS and HPLC/ESI-MSn provided complementary information for the identification of the constituents in Dan-shen. Forty constituents were identified in 30 min based on their positive and negative ion ESI mass spectra and liquid chromatographic information. Thus the method described is useful for the rapid analysis of multiple constituents in Dan-shen.     

Classification of diterpenoid alkaloids from Aconitum kusnezoffii Reichb. by liquid chromatography-tandem mass spectrometry-based on molecular networking

Trace amounts of components in traditional Chinese medicine are considered pharmacological active substances used for treating many serious diseases. However, purifying all the trace substances and making clear their structures are not easy. In this context, high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry based molecular networking was applied to investigate the chemical constituents of the roots of Aconitum kusnezoffii Reichb., which led to the identification of 33 nodes in different groups ( N1 – N33 ). Based on the excremental fragmentation pathway of known diterpenoid alkaloids ( 1 – 9 ) and comparisons of characteristic ions and characteristic loss of analogs in literature, the structures of unknown ions were deduced. This work lays a foundation for the evaluation of the clinical basis and mechanism of traditional Chinese medicine from the aspects of chemistry. In this paper, the method speculation of unknown natural products by means of molecular network method is expected to be applied in the discovery and change law of relevant active components in clinical pharmacology and the change of complex systems caused by trace active compounds.     

Characterization of compounds from the roots of Saposhnikovia divaricata by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

In this study, four types of compounds including coumarins, chromones, furoylmethyl amino acid derivative and benzofuran glycoside were isolated from the roots of Saposhnikovia divaricata. The electrospray ionization (ESI) mass spectral fragmentation pathways of these compounds were proposed. In particular, the ESI-MS(n) fragmentation behavior of linear dihydrofurocoumarins, dihydrofuro- and dihydropyranochromones were deduced in detail. For the linear dihydrofurocoumarins, the fragmentation was triggered by the initial loss of the C-4' substituting group. Then, the characteristic ions were observed followed by the losses of 15, 18, 28 and 46 Da. It is noteworthy that the elimination of H(2)O (18 Da) from the cleavage of the dihydrofuran ring is reported for the first time. For the linear dihydrofurochromones, characteristic eliminations of 18, 48 and 72 Da were observed. The loss of 18 Da could arise from two different fragmentation pathways, and the observed ion was composed of a mixture of two different structural ions. For the linear dihydropyranochromones, it was found that the dihydropyran ring was converted into the pyran ring by the elimination of the C-3' substituting group. This fragmentation was followed by the diagnostic losses of 18, 28, 42 and 54 Da in tandem mass spectrometry. The above fragmentation rules were successfully applied for the analysis of the chemical constituents of the roots of Saposhnikovia divaricata. A total of 32 compounds were identified or tentatively characterized by HPLC/DAD/ESI-MS(n). Among them, eight compounds were new and seven compounds were reported from that genus for the first time.     

Purification and preparation of compounds from an extract of Scutellaria barbata D. Don using preparative parallel high performance liquid chromatography

Preparative parallel high performance liquid chromatography combined with solvent partition and other pretreatments were adopted to separate and purify compounds from an extract of Scutellaria barbata D. Don. Mass-triggered fraction collection allowed the rapid and precise isolation of target compounds. Twelve compounds were isolated from the extract of S. barbata D. Don, their purity in area percent was determined by HPLC analysis, and the structures of seven compounds were further identified with HPLC/ESI-MS, (1)H NMR, and( 13)C NMR, among which 4-(3,4-dihydroxy-phenyl)-but-3-en-2-one, acacetin-7-diglucuronide, and luteolin-7-diglucuronide were the first to be identified from this plant. The results demonstrated that multi-channel parallel preparative HPLC/UV/MS is an efficient method for isolation and purification of compounds from natural products.     

青海翠雀化学成分的研究

用柱层析、制备薄层层析等对青海翠雀的化学成份进行了分离，并用IR，FA， MS，1HNMR，13C NMR及DEPT等光谱确定了化合物的结构，共分得5种二萜生物碱， 其中一种为新化合物，还分得一种非生物碱β-谷甾醇．     

Rapid structural characterization of triterpenoid saponins in crude extract from Symplocos chinensis using liquid chromatography combined with electrospray ionization tandem mass spectrometry

Triterpenoid saponins in bioactive crude extract from Symplocos chinensis were rapidly identified using electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and liquid chromatography coupled with sequential mass spectrometry (LC-MSn). According to the characteristic fragmentation behavior of known glucuronide-type triterpenoid saponins isolated from this plant, a total of fourteen constituents in the crude extract were structurally characterized on the basis of their retention time and tandem mass spectrometric analysis, including five pairs of naturally occurring isomers. Except one known saponin formerly obtained, the other constituents were new compounds. The analytical method of LC-MSn combined with ESI-MSn in positive and negative ion modes has been developed for the direct structural elucidation of triterpenoid saponins of this kind in plant extracts.     

Rapid structural characterization of isomeric benzo[c]phenanthridine alkaloids from the roots of Zanthoxylum nitidium by liquid chromatography combined with electrospray ionization tandem mass spectrometry

The fragmentation mechanism of six alkaloids, namely: dihydronitidine, dihydrochelerythrine, 8-acetonyldihydronitidine, 8-acetonyldrochelerythrine, nitidine and 1,3-bis(8-dihydronitidinyl)acetone, was investigated by electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn). Tandem mass spectrometry experiments indicated that different substitution sites of the methoxyl groups at C-9 and C-10 or at C-10 and C-11 determined the different abundances of the MS2 fragmentation ions using the same collision energy. According to the different abundances of MS2 product ions, positional isomeric benzo[c]phenanthridine alkaloids can be differentiated. Moreover, ten constituents in the crude alkaloidol extract from the roots of Zanthoxylum nitidium were rapidly identified by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC/MSn), through comparing the retention times and ESI-MSn spectra with the authentic standards. This work demonstrates that not only the characteristic fragments but also the characteristic abundances of the fragment ions can be used for detailed structural characterization.     

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.     

Identification of C-21 steroidal glycosides from the roots of Cynanchum chekiangense by high-performance liquid chromatography/tandem mass spectrometry

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) was used to identify C-21 steroidal glycosides with immunological activities in roots of Cynanchum chekiangense. In the MS/MS spectra, fragmentation reactions of the [M + Na]⁺ were recorded to provide structural information about the glycosyl and aglycone moieties. To further confirm the fragments structures, off-line Fourier transform ion cyclotron resonance tandem mass spectrometry (FT-ICR-MS/MS) was also performed. In the study, four known steroidal glycosides cynascyroside C, chekiangensosides A and B, glaucoside H, and four novel steroidal glycosides chekiangensosides C, D, E and chekiangensoside A isomer were identified based on mass spectral data, NMR spectral data and standards. This is the first report on identifying steroidal glycosides in roots of C. chekiangense by HPLC/ESI-MS/MS directly, which could save time and material consuming efforts in traditional phytochemistry analysis.     

Mass spectrometrically detected directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry for the identification of xenobiotic metabolites in maize plants

Reconstructed ion chromatograms have been used to identify relevant high performance liquid chromatography (HPLC) peaks in a directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry (HPLC/NMR/MS) experiment. This has been applied to a study of the metabolism of a model compound, 5-nitropyridone (2-hydroxy-5-nitropyridine), in maize plants grown hydroponically. By monitoring the on-flow reconstructed ion chromatogram corresponding to the 5-nitropyridone fragment at m/z 143, and additional molecular ions corresponding to metabolites identified as products from similar compounds, relevant peaks were identified rapidly for subsequent stopped-flow 1H NMR spectroscopic analysis. The combination of coupled HPLC/NMR/MS enabled the direct identification of three metabolites, namely the N-glucoside, N-malonylglucoside, and O-malonylglucoside. This work demonstrates the power of HPLC/NMR/MS for the structural elucidation of xenobiotic metabolites in complex biological matrices (such as plant material) with minimal sample preparation. In particular, using mass spectrometry for the initial identification of relevant HPLC peaks allows the analysis of complex samples without the necessity for other spectroscopic markers, such as 19F NMR signal for fluorinated compounds or UV spectroscopy for molecules with strong UV chromophores.     

Multistage electrospray ionization mass spectrometric analyses of sulfur-containing iridoid glucosides in Paederia scandens

Paederia scandens is a traditional Chinese herbal medicine that possesses important bioactive sulfur-containing iridoid glucosides. This study reports the investigation of the fragmentation behavior of four sulfur-containing iridoid glucosides isolated from P. scandens by ion trap electrospray ionization tandem mass spectrometry (ESI-MSn). Both multistage electrospray ionization (ESI-MSn) experiments in an ion trap instrument and high-resolution and accurate mass measurement in an ESI-Q-TOF mass spectrometer in positive mode were used to elucidate the main fragmentation pathways of these compounds. These experiments yielded essentially the same product ions in both ion trap and Q-TOF instruments, and their fragmentation patterns were found useful for their characterization, which were applied to elucidate a trace compound in the extracts of P. scandens by on-line LC/ESI-MSn. Major and diagnostic product ions have been identified and their origins are proposed.     

High-performance liquid chromatography-diode array detection/electrospray ionization mass spectrometry for the simultaneous analysis of cis-, trans- and dihydro-2-glucosyloxycinnamic acid derivatives from Dendrobium medicinal plants

Sensitive, selective and reliable high-performance liquid chromatography (HPLC)-diode array detection (DAD)/electrospray ionization multi-stage mass spectrometry (ESI-MSn) methods have been developed for the characterization of nine 2-glucosyloxycinnamic acid derivatives and quantitative analysis of three of the major 2-glucosyloxycinnamic acids, cis-melilotoside, trans-melilotoside and dihydromelilotoside, present in Dendrobium medicinal plants. The identities of the latter three major 2-glucosyloxycinnamic acids were confirmed by comparing their retention times, UV and mass spectra with those of the reference standards. The characteristic ESI-MSn fragmentation patterns of the remaining six 2-glucosyloxycinnamic acid derivatives, which are similar to the three major compounds, have allowed the putative elucidation of their structures. The concentrations of the cis-, trans- and dihydromelilotosides were simultaneously determined by HPLC/ESI-MS2 using the multiple reaction monitoring (MRM) mode in extracts of Dendrobium species. The method was validated with respect to the overall intra- and inter-day variation (RSD less than 8%) and the limits of quantification for the cis-, trans- and dihydromelilotosides were 0.09, 0.09 and 0.01 microg/mL, respectively.     

New halogenated furanones from the marine alga delisea pulchra (cf. fimbriata)

An investigation of the natural products chemistry of the red alga Delisea pulchra, collected from the Cape Banks, New South Wales, Australia, yielded eight new polyhalogenated furanones (1–7) and the previously reported metabolites 8–24). The structures of 1–8 were determined from the interpretation of their 1D and 2D NMR, UV, IR and mass spectral data. For the first time, complete ¹H and ¹³C NMR data for compounds 14, 18, and 20-23 are reported.     

Nuclear magnetic resonance and liquid chromatography-mass spectrometry combined with an incompleted separation strategy for identifying the natural products in crude extract

NMR and LC-MS combined with an incompleted separation strategy were proposed to the simultaneous structure identification of natural products in crude extracts, and a novel method termed as NMR/LC-MS parallel dynamic spectroscopy (NMR/LC-MS PDS) was developed to discover the intrinsic correlation between retention time (Rt), mass/charge (m/z) and chemical shift (δ) data of the same constituent from mixture spectra by the co-analysis of parallelly visualized multispectroscopic datasets from LC-MS and ¹H NMR. The extracted ion chromatogram (XIC) and ¹H NMR signals deriving from the same individual constituent were correlated through fraction ranges and intensity changing profiles in NMR/LC-MS PDS spectrum due to the signal amplitude co-variation resulted from the concentration variation of constituents in a series of incompletely separated fractions. NMR/LC-MS PDS was applied to identify 12 constituents in an active herbal extract including flavonol glycosides, which was separated into a series of fractions by flash column chromatography. The complementary spectral information of the same individual constituent in the crude extract was discovered simultaneously from mixture spectra. Especially, two groups of co-eluted isomers were identified successfully. The results demonstrated that NMR/LC-MS PDS combined with the incompleted separation strategy achieved the similar function of on-line LC-NMR-MS analysis in off-line mode and had the potential for simplifying and accelerating the analytical routes for structure identification of constituents in herbs or their active extracts.     

Rapid identification of C21 steroidal saponins in Cynanchum versicolor Bunge by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and liquid chromatography coupled with on-line electrospray ionization tandem mass spectrometry (LC/ESI-MSn) were performed to elucidate the clearage rule of nine investigated C21 steroidal saponins and identify them in the saponin fraction of 90% ethanolic extracts from the root and rhizome of Cynanchum versicolor Bunge. The fragments of C21 steroidal saponins in positive and negative ESI-MSn were used to deduce their mass spectral fragmentation mechanisms, and their structures were further confirmed by ESI-MSn in positive mode. The MSn spectra of the [M+Na]+ ions for saponins provided a wealth of structural information on glycosidic bond cleavage, which allowed a straightforward interpretation of spectra, with respect to the identifications of features such as the sequences of sugars attached to saponins and sugar type. By using LC/ESI-MSn, nine C21 steroidal saponins were detected in the saponin fraction of C. versicolor, and an isomer of atratoglaucoside A was elucidated simultaneously. All nine compounds showed an abundant ion for the loss of 46 Da (HCOOH) from [M+Na]+. The losses of monosaccharide sequences and aglycone as neutral fragmentation from [M+Na-HCOOH]+ were also acquired as the characteristic ions of these C21 steroidal saponins. It provided important information on monosaccharide sequences and in particular on sugar types and could be used to identify and elucidate other C21 steroidal saponins. These studies allowed us to rapidly identify C21 steroidal saponins from Radix cynanchi atrati. It is indicated that the described method had wide applicability to rapidly screen and provide structural confirmation on C21 steroidal saponins in crude materials.     

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.     

Rapid structural determination of amides in Piper longum by high-performance liquid chromatography combined with ion trap mass spectrometry

A reversed-phase liquid chromatography/tandem multistage mass spectrometry (MS/MS) method was developed for the characterization of amides from the extracts of Piper longum. The characteristic fragmentations of the amides found in P. longum showed diagnostic structural information. Extracted ion chromatography (EIC) and constant neutral losses were used to guide the search for the amides of interest. Amides of known structures that contain four subtypes of amides were rapidly determined, and novel amides were also identified for this plant. Forty-two amides were rapidly identified, of which 22 were found in this plant for the first time and 9 were new compounds. The method is convenient and sensitive, especially for minor components in the unpurified, complex mixture; the structures of unknown constituents could be determined, in the absence of authentic sample, by comparison of the fragmentation patterns with those of homologous compounds.     

Novel antimycobacterial benzoxazole alkaloids, from the west Indian Sea whip Pseudopterogorgia elisabethae

[formula: see text] Our screening for marine natural products with anti-tuberculosis activity from the West Indian gorgonian coral Pseudopterogorgia elisabethae resulted in the isolation of two active diterpenoid alkaloids, pseudopteroxazole (1) and seco-pseudopteroxazole (2). Their structures were elucidated by NMR spectral analysis, including a variety of two-dimensional techniques. Compounds 1 and 2 are previously undescribed diterpenoids containing the uncommon benzoxazole moiety. Biological screening studies indicated that pseudopteroxazole (1) is a potent growth inhibitor of Mycobacterium tuberculosis H37Rv, while seco-pseudopteroxazole (2) shows moderate to strong inhibitorial activity.     

Electrospray ionization tandem mass spectral analysis of oxidation products of precursors of sulfur mustards

Electrospray ionization tandem mass spectral (ESI-MSn) analysis of thiodiglycol, bis(2-hydroxyethylthio)alkanes (BHETAs) and their mono-, di-, tri-, and tetraoxygenated compounds was carried out to obtain their characteristic spectra for ESI-MS analysis. These compounds are important markers of chemical warfare agents, namely sulfur mustards. ESI-MSn (n > or = 3) analysis of a compound by collisionally induced dissociation in an ion trap gives rise to mass spectra that are somewhat similar to electron ionization mass spectra. These ESI-MSn spectra can be used for compound identification. Under ESI-MS and ESI-MS/MS the compounds mostly produced [M+NH4]+, [M+H]+ and [M+H--H2O]+ ions. Fragmentations of these even-electron precursors in the ion trap gave rise to characteristic product ions via neutral loss of O2, H2O, C2H4, HCHO, C2H4O, C2H4S, HSC2H4OH and C2H4SO. Fragmentation routes of these compounds are proposed that rationalize the formation of product ions in ESI-MSn analysis.