Structural characterization and identification of dibenzocyclooctadiene lignans in Fructus Schisandrae using electrospray ionization ion trap multiple-stage tandem mass spectrometry and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry

The electrospray ionization ion trap multiple-stage tandem mass spectrometry (ESI-MSⁿ) and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-ICR-MSⁿ) have been applied successfully to the direct investigation of a number of dibenzocyclooctadiene lignan constituents from the methanol extracts of the Fructus Schisandrae in the positive ion mode. The detailed structural characterization of the same skeleton and different peripheral substituents had been studied and the precise elemental compositions of ions at high mass resolution had been obtained. So the fragmentation mechanisms could be clarified. And the lignan components in Schisandra chinensis (Turcz.) Baill. fruits (SCF) and Schisandra sphenanthera Rehd. et Wils. fruits (SSF) were identified by comparing the structural information and fragmentation mechanisms. Then a pair of isobaric compounds was differentiated. Meanwhile these two similar fruits were distinguished. The research results demonstrated that ESI-MSⁿ technique is a sensitive, selective and effective tool for the direct analysis and rapid determination of constituents in complex mixtures from nature products. And these should be useful for the identification of similar compounds and differentiation of similar species from Chinese herbs.     

Structural analysis of monoterpene glycosides extracted from Paeonia lactiflora Pall. using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Paeoniflorin standard was first investigated by electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) using a sustained off-resonance irradiation (SORI) collision-induced dissociation (CID) method at high mass resolution. The experimental results demonstrated that the unambiguous elemental composition of product ions can be obtained at high mass resolution. Comparing MS/MS spectra and the experimental methods of hydrogen and deuterium exchange, the logical fragmentation pathways of paeoniflorin have been proposed. Then, the extracts of the traditional Chinese medicine Paeonia lactiflora Pall. were analyzed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). By comparison with the ESI-FTICR-MS/MS data of paeoniflorin, the isomers paeoniflorin and albiflorin in Paeonia lactiflora Pall. have been identified using HPLC/MS with CID in an ion trap and in-source CID. Furthermore, using the characteristic fragmentation pathways, the retention times (t(R)) in HPLC and MS/MS spectra, the structures of three other kinds of monoterpene glycoside compounds have been identified on-line without time-consuming isolation. Thus an HPLC/ESI-MS method for the analysis of constituents in Paeonia lactiflora Pall. has been established.     

On-line identification of phenanthroindolizidine alkaloids in a crude extract from Tylophora atrofolliculata by liquid chromatography combined with tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with sequential mass spectrometry (LC/MS(n)) were applied to identify trace-level phenanthroindolizidine alkaloids in crude extracts from Tylophora atrofolliculata. Based on the relationship between the characteristic fragmentation reactions and the structural features of related compounds of known structure from this plant, the bioactive crude extract was analyzed in detail by positive and negative ion ESI-MS(n), LC/UV-MS and LC/MS(n) techniques. A total of nine constituents in the crude extract were identified rapidly, including several isomers; seven of these constituents are new and two are known compounds. The structures of four of these constituents were subsequently confirmed by nuclear magnetic resonance (NMR) and accurate mass measurements using high-resolution fast-atom bombardment mass spectrometry (FAB-HRMS).     

Studies on principal components and antioxidant activity of different Radix Astragali samples using high-performance liquid chromatography/electrospray ionization multiple-stage tandem mass spectrometry

The principal components, isoflavonoids and astragalosides, in the extract of Radix Astragali were detected by a high-performance liquid chromatography couple to electrospray ionization ion trap multiple-stage tandem mass spectrometry (HPLC-ESI-IT-MSⁿ) method. By comparing the retention time (t_R) of HPLC, the ESI-MSⁿ data and the structures of analyzed compounds with the data of reference compounds and in the literature, 17 isoflavonoids and 12 astragalosides have been identified or tentatively deduced. By virtue of the extracted ion chromatogram (EIC) mode, simultaneous determination of isoflavonoids and astragalosides could be achieved when the different components formed overlapped peaks. And this method has been utilized to analyze the constituents in extracts of Radix Astragali from Helong City and of different growth years. Then the antioxidant activity of different samples has been successfully investigated by HPLC-ESI-MS method in multiple selected ion monitoring (MIM) mode, applying the spin trapping technology, and the Ferric Reducing Antioxidant Power (FRAP) assay was applied to support the result. The correlations of the isoflavonoids and astragalosides components and the antioxidant activities of Radix Astragali were summarized. The present paper demonstrates that HPLC-ESI-MSⁿ is a powerful method for the characterization of the principal components and evaluation of the antioxidant activity of Chinese medicinal herbs.     

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.     

Detection, characterization and identification of phenolic acids in Danshen using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry

By using HPLC-diode array detection-electrospray ion trap tandem mass spectrometry (HPLC-DAD-ESI-MS(n)) in negative ion mode, we have analyzed the fragmentation pathways of 11 phenolic acids which were isolated from Danshen. Then the extract of Danshen was analyzed, and a total of 42 phenolic acids, including sixteen new minor constituents, were identified or tentatively identified for the first time. A new solid-phase extraction (SPE) method, new HPLC separation method, new liquid chromatography (LC)-MS and LC-MS(n) (n=3-5) data and proposed fragmentation pathways, LC retention time for phenolic acids are reported.     

Isolation and characterization of flavonols from blackcurrant by high-performance counter-current chromatography and electrospray ionization tandem mass spectrometry

Blackcurrant is considered as a natural high-value food raw material and possesses a variety of therapeutic properties. The health benefits of blackcurrant have generally been credited to its high anthocyanin content; however, the therapeutic properties of other minor flavonoids constituents have not yet been investigated due the difficulties related to their isolation. Multiple steps of high-performance counter-current chromatography in combination with ESI tandem mass spectrometry (MS(n)) were successfully used for the preparative isolation of flavonols from blackcurrant extract, to study their electrospray ionization mass spectrometry fragmentation behavior. Seven flavonols, namely myricetin-3-O-rutinoside (145.5 mg), myricetin-3-O-hexoside (79.7 mg), myricetin-3-O-(6″-malonyl)-glucoside (17.4 mg), kaempferol-3-O-glucoside (20.5 mg), quercetin-3-O-rutinoside (55.1 mg), quercetin-3-O-hexoside (25.8 mg), and myricetin (129.1 mg) have been successfully isolated and their multistage MS(n) data were used for detailed structure characterization. The results of these experiments demonstrated that high-performance counter-current chromatography along with ESI-MS(n) is a sensitive, selective, and effective technology for isolation and characterization of minor constituents from a complex mixture.     

Sequencing of new beauverolides by high-performance liquid chromatography and mass spectrometry.

Mass spectrometry (MS) and tandem mass spectrometry (MS(n)) were used for the identification of beauverolides in the fermentation broth of Beauveria bassiana and for evaluation of the purified fraction obtained by sublimation of beauverolides. Besides being a new efficient route for purification of beauverolides, sublimation provided an enrichment of new minor lipophilic beauverolides of lower molecular weight from the original complex mycelial extract. The product ion collision-induced dissociation (CID) spectra obtained on an ion trap (electrospray ionization), the in-source CID mass spectra on a sector instrument (atmospheric-pressure chemical ionization) and the post-source decay matrix-assisted laser desorption/ionization mass spectra of beauverolides were compared and evaluated. All MS(n) experiments started with singly charged precursor ions. The following two new representatives of this group of compounds were identified by high-performance liquid chromatography and MS (HPLC/MS): cyclo-(3-hydroxy-4-methyloctanoyl-valyl-alanyl-leucyl) and cyclo-(3-hydroxy-4-methyloctanoyl-tyrosyl-alanyl-leucyl). Individual structures were confirmed by preparative isolation and nuclear magnetic resonance spectroscopy. The structure of a third novel and minor beauverolide was tentatively assigned by HPLC/MS only as cyclo-(3-hydroxy-4-methyldecanoyl-valyl-alanyl-Lxx), Lxx = leucyl, isoleucyl, or allo-isoleucyl.     

Negative electrospray ionization tandem mass spectrometric investigation of ent-kaurane diterpenoids from the genus Isodon

ent-Kaurane diterpenoids are a class of natural compounds isolated from genus Isodon, which have been found to have important bioactivities. Negative electrospray ionization tandem mass spectrometry ((-)ESI-MS(n)) was used to investigate the fragmentation pattern of C-20-nonoxygenated ent-kauranes and two subtypes of C-20-oxygenated ent-kauranes by using an ion trap instrument and accurate mass measurement on an ESI-Q-time-of-flight (TOF) mass spectrometer. The analysis revealed that loss of CH(2)O or CO(2) is the predominant process for 7, 20-epoxy and 7, 20 : 14, 20-diepoxy subgroup of C-20-oxygenated ent-kauranes. In addition, compounds of C-20-nonoxygenated ent-kauranes with a conserved core structure but different substituent groups, such as a hydroxyl, aldehyde, carboxyl, and acetyl moiety, resulted in diagnostic product ions through losses of H(2)O, CO, CO(2), and AcOH, respectively. This work clearly demonstrates the utility of tandem mass spectrometry for studies on the rationalization of the diagnostic fragmentation of a series of compounds from two main types of the ent-kaurane diterpenoids.     

Identification of metabolites in WZS‐miniature pig urine after oral administration of Danshen decoction by HPLC coupled with diode array detection with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry

Danshen (DS) is a widely used traditional Chinese medicine for treating cardiovascular and cerebrovascular diseases. A simple, rapid and sensitive method was developed for identification of the in vivo metabolites in urine of WZS‐miniature pigs after oral administration of DS decoction by HPLC coupled with diode array detection with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry. This method has been successfully applied to simultaneous identification of 50 compounds (including 11 new ones) in pig urine. In addition, one new compound, (3‐hydroxyphenyl) crylic acid glycine methyl ester (C1), along with eight known ones were first isolated by column chromatography and identified by spectroscopic means, including 1D/2DNMR and mass spectrometry, as reference substances. Ten phenolic compounds (protocatechuic aldehyde, protocatechuic acid, caffeic acid, danshensu, ferulic acid, isoferulic acid, rosmarinic acid and salvianolic acid A/B/D) were found to be the main absorbed original constituents of DS decoction, which underwent the metabolic reactions of glucuronidation, sulfation, methylation, hydrogenation and glycine conjugation in vivo. In conclusion, the developed method is applicable to the analysis and identification of constituents in biological matrices after administration of DS decoction. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of three major triterpenoids in epicuticular wax of cabbage (Brassica oleracea L.) by high-performance liquid chromatography with UV and mass spectrometric detection

Lupeol, together with alpha- and beta-amyrins in smaller quantities, has been found for the first time in the epicuticular wax of white cabbage (Brassica oleracea L. convar. capitata (L.) Alef. var. alba DC) leaf surface extract. The three triterpenoids were identified by a new high-performance liquid chromatographic (HPLC) method with UV and mass spectrometric (MS) detection using atmospheric pressure chemical ionization (APCI). All three isomeric compounds gave a parent ion peak at m/z 409 [M+H-18](+) and the relative intensities of some characteristic fragment ion peaks in tandem mass spectrometric (MS-MS) spectra of this parent ion enabled differentiation between the isomers. An additional peak at m/z 439 [M+H](+), which could be oleanonic or ursonic aldehyde, was detected by HPLC-APCI-MS. Saponification of cabbage leaf surface extract with 20% NaOH in methanol at 65 degrees C for 2h had no influence on lupeol, or alpha- or beta-amyrins, but lead to the formation of three additional compounds, which were not identified.     

Analysis of the volatile compounds in Senecio scandens Buch-Ham by gas chromatography-tandem mass spectrometry based on diversified scan technologies

Static headspace gas chromatography-tandem mass spectrometry was used to identify volatile compounds from Senecio scandens Buch-Ham. The elemental composition of compounds was confirmed by exploiting the tandem mass spectra of isotopic peaks from the precursor ion. Some isomers were well distinguished by the diversified scan technologies of tandem mass spectrometry (MS/MS). The MS/MS included a product ion scan, a precursor ion scan and a neutral loss scan. The results showed that 46 volatile compounds were completely identified, and the great of majority compounds were α-pinene (11.93%), n-caproaldehyde (9.02%) and dehydrosabinene (6.22%). This qualitative method is convenient and accurate and can be considered as a complementary identification method for the qualitative analysis of volatile compounds in complex samples.     

Characterization ofC-glycosylflavones fromDissotis rotundifolia by liquid chromatography — UV diode array detection — tandem mass spectrometry

Summary By high-performance liquid chromatography (HPLC) coupled with UV diode-array detection (DAD) and thermospray mass spectrometry (TSP-MS), four main constituents of a polar, whole plant extract fromDissotis rotundifolia T. were characterized. The fourC-glycosylflavones, isoorientin, orientin, vitexin and isovitexin were detected in the methanolic and hydroalcoholic extract of the plant as well as in the commercial drug preparation ‘Sirop de Dissotis’. Although the UV data and TSP mass spectra allowed rapid characterisation of all fourC-glycosylflavones, exact attribution of the peaks to their structures could not be achieved as neither the UV spectra nor the TSP mass spectra enabled differentiation of one position isomer from the other. Therefore a successful attempt was made to distinguish the 6-C from the 8-C-glycosylflavones by thermospray tandem mass spectrometry (TSP-MS-MS). The collision induced dissociation (CID) spectra of the particular ion [M+H-120]⁺ gave fragments which permitted differentiation of position isomers. To confirm the accuracy of on-line results, reference compounds were included in the HPLC study.     

Determination of linkage position and anomeric configuration in Hex-Fuc disaccharides using electrospray ionization tandem mass spectrometry

Fixed-energy sequential tandem mass spectrometry (MS(n)) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of six isomers of Gal-Fucalpha-OBenzyl disaccharides. Under collision-induced dissociation (CID), sodiated molecular species generated in the positive-ion electrospray ionization mode yield simple and predictable mass spectra. Information on interglycosidic linkages and configurations can be deduced from the relative intensities of the selected diagnostic fragments arising from the glycosidic bond cleavages and corroborated by the fragments arising from cross-ring cleavages. As the CID patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by matching the spectra with a library. The rules governing the fragmentation behavior of this class of oligosaccharides were tested for a representative isomeric disaccharide, Glcbeta1,3Fucalpha-OAllyl. The findings establish a basis for using MS(n) with a quadrupole ion trap instrument to elucidate structures of hexose-fucose subunits from more complicated oligosaccharides. Energy-resolved mass spectra were also acquired by CID tandem triple-quadrupole mass spectrometry. The breakdown behavior of the molecular ions revealed patterns which could differentiate stereoisomers of Gal-Fuc disaccharides over a range of collision energy from 20 to 50 eV.     

Systematic screening and characterization of the major bioactive components of Poria cocos and their metabolites in rats by LC-ESI-MS(n)

Poria cocos is a well-known medicinal plant widely used in China and other East Asian countries owing to its various therapeutic effects. However, the bioactive constituents responsible for the pharmacological effects of Poria cocos and their metabolites in vivo are still unclear to date. The aim of the present study was to develop a practical method based on the combined use of the liquid chromatography coupled with electrospray ionization multistage tandem mass spectrometry (LC-ESI-MS(n) ) for the comprehensive and systematic separation and characterization of the bioactive constituents of Poria cocos extract and their metabolites in rats. Based on the proposed strategy, a total of 34 compounds were characterized from the extract of Poria cocos. Among them, eight were unambiguously identified by comparing their retention times and mass spectra with those of reference standards, and 26 were tentatively identified on the basis of their MS(n) fragmentation behaviors and molecular weight information from literatures. In vivo, seven compounds were successfully detected in rat urine whereas one was found in rat plasma. This study proposed a series of potential bioactive components and provided helpful chemical information for further research on the action mechanism of traditional Chinese medicine.     

Qualitative screening of phenolic compounds in olive leaf extracts by hyphenated liquid chromatography and preliminary evaluation of cytotoxic activity against human breast cancer cells

In this work, high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap multiple-stage tandem mass spectrometry (ESI-IT-MS²) has been applied to screen phenolic compounds in olive leaf extracts. The use of a small particle size C18 column (1.8 μm) provided great resolution and made separation of a lot of isomers possible. The structural characterization was based on accurate mass data obtained by ESI-TOF-MS, and the nature of fragmentation ions were further confirmed by ESI-IT-MS² when possible. In addition, we employed tetrazolium salt (MTT)-based assays to assess the effects of olive leaf extracts on the growth of human tumor-derived cells. Upon this approach, we achieved an accurate profile of olive leaf phenolics along with the identification of several important isomers of secoiridoids and flavonoids. This will allow a better understanding of the complete composition of olive-leaf-bioactive compounds as well as their involvement in Olea europaea L. biochemical pathways. Importantly, olive leaf extracts exhibited dose-dependent inhibitory effects on the metabolic status (cell viability) of three breast cancer models in vitro. Since the tumoricidal activity of the extracts should be mainly attributed to the identified olive leaf phenolics, these findings warrant further investigation at the structure–function molecular level to definitely establish the anticancer value of these phytochemicals.     

ESI-QqTOF-MS/MS and APCI-IT-MS/MS analysis of steroid saponins from the rhizomes of Dioscorea panthaica

Using high-resolution quadrupole time-of-flight mass spectrometry along with an electrospray ionization source (ESI-QqTOF-MS), accurate molecular weights of 13 steroid saponins extracted from the rhizomes of Dioscorea panthaica were acquired and the corresponding molecular formulae obtained. In order to elucidate the fragmentation pathways of steroid saponins in D. panthaica, 10 authentic samples were investigated using ESI-QqTOF-MS/MS. In addition, atmospheric pressure chemical ionization mass spectrometry combined with ion trap tandem mass spectrometry (APCI-IT-MS/MS) was used to analyze the structures of 13 steroid saponins in D. panthaica. Through the analysis of their tandem mass data, diagnostic fragment ions of the spirostanol and furostanol steroid saponins in D. panthaica were detected as m/z 271.2056 and 253.1951. In addition, four pairs of isomers were detected and the possible structures of four unknown steroid saponins in D. panthaica speculated. ESI-TOF and APCI-MS(n) have proved to be effective tools for research on fragmentation mechanism of steroid saponins and the rapid determination of native steroid saponins in extract mixture, thereby avoiding tedious derivation and separation steps.     

Quality evaluation of cortex moutan by high performance liquid chromatography coupled with diode array detector and electrospary ionization tandem mass spectrometry

An high performance liquid chromatography (HPLC) coupled with diode array detector (DAD) and electrospray ionization tandem mass spectrometry (ESI/MS(n)) method was developed for quality evaluation of Cortex Moutan through identification of common constituents based on chromatographic fingerprints and determination of key pharmacological compounds. The representative chromatographic fingerprints of Cortex Moutan were obtained by analyzing 10 batches of samples under the optimized HPLC conditions and the results showed that the chromatographic profiles of the analyzed samples were very similar. Total of nineteen common peaks were detected and seventeen of them were identified rapidly by their characteristic UV profile and the information of molecular structure provided by ESI/MS(n) experiments. Simultaneously, five key pharmacological compounds, namely gallic acid, oxypaeoniflorin, paeoniflorin, benzoylpaeoniflorin and paeonol, were determined by the validated HPLC-DAD method. The linear calibration curves were acquired with correlation coefficients higher than 0.999. The precisions of intra-day and inter-day were not exceeding 3.1%, and the recoveries of five analytes were from 92.86 to 99.35%. This developed method that combined the chromatographic fingerprints and quantification assay ensured the phytoequivalence and pharmacological effects of Cortex Moutan and was successfully applied to the quality control of Cortex Moutan.     

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.     

In vivo microdialysis and reverse phase ion pair liquid chromatography/tandem mass spectrometry for the determination and identification of acetylcholine and related compounds in rat brain

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. A microdialysis probe was surgically implanted into the striatum of the rats and Ringer's solution was used as the perfusion medium at a flow rate of 2 microL per minute. The samples were then analyzed off-line by LC/MS/MS experiments. The separation of ACh and choline (Ch) was carried out using reverse phase ion pair liquid chromatography with heptafluorobutyric acid as a volatile ion pairing reagent. Analytes were detected by electrospray ionization tandem mass spectrometry in the positive ion mode. The detection limit for ACh was 1.4 fmol on column, which is at least three times lower than previously reported. Three quaternary ammonium compounds in the rat brain microdialysate were also identified by tandem mass spectrometry experiments in which the unknown mass spectra were compared with standard reference compounds. These compounds were identified as carnitine, acetylcarnitine and (3-carboxypropyl)trimethylammonium. This is the first known report of the compound (3-carboxypropyl)trimethylammonium being found in rat brain.