Rapid structural determination of modified pregnane glycosides from Cynanchum forrestii by liquid chromatography-diode-array detection/electrospray ionization multi-stage tandem mass spectrometry

The fragmentation behaviors of the two types of modified pregnane glycosides from Cynanchum forrestii were investigated by positive ion electrospray ionization multi-stage tandem mass spectrometry equipped with an ion trap analyzer. The spectral data further illuminated the predominance of ESI-MSⁿ technique on the identification of pregnane glycosides, especially of two sorts of modified pregnane glycosides with aglycone skeletons of 13,14:14,15-disecopregnane-type and 14,15-secopregnane-type, which differed in the presence of the characteristic [M−46+Na]⁺ ion. For sugar residues, the fragment ions were analyzed and some possible fragmentation pathways were proposed, especially for 3-demethyl-2-deoxythevetose, the glycosidic cleavage reaction was easier to occur than those of other sugar units in its moiety. The natures and differences of the pregnane cores, and the types and linked sequences of sugar residues were illustrated. According to these conclusions, eight new pregnane glycosides in the fraction of Cynanchum forrestii were structurally elucidated by HPLC/HRMS and HPLC-DAD/ESI-MSⁿ techniques. Results of the present studies can benefit the rapid identification and structural determination of analogous constituents in crude plant extracts.     

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.     

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.     

A study of the analytical behaviour of selected psycho-active drugs using liquid chromatography, ion trap mass spectrometry, gas chromatography and polarography and the construction of an appropriate database for drug characterisation

This paper provides analytical chemical information on a range of psycho-active drugs. This analytical chemical information on liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS), ion trap mass spectrometry (ESI-MSⁿ), gas chromatography-flame ionisation detection (GLC-FID) and polarographic behaviour is then incorporated into a database which is of use in drug characterisation. Application is found in the determination of selected drug compounds in hair samples.     

Studies on the Components of Crude and Processed Fructus Corni by ESI-MS

The components of crude and processed Fructus Corni were investigated by means of electrospray ionization-tandem mass spectrometry（ESI-MSn） technique in the negative ion mode. Compared with those of crude Fructus Corni, the chemical components of the processed Fructus Corni were changed both in quality and in quantity. From the ESI-MS spectra of the crude and processed Fructus Corni, six peaks were selected to establish the characte-ristic ESI-MS peaks. Several factors in the processing procedure were examined. The experimental results demonstrate that the chemical reactions that occurred in the processing procedure can be used for the elucidation of the processed mechanism of Fructus Corni, which is regularly affected by the processing conditions. The present article provides both the chemistry evidence for the understanding of the processing procedure of Fructus Corni and the specific methodology for the research of the processing procedure and quality identification of traditional Chinese medicine.     

A study of the analytical behaviour of selected synthetic and naturally occurring quinolines using electrospray ionisation ion trap mass spectrometry, liquid chromatography and gas chromatography and the construction of an appropriate database for quinoline characterisation

Mass spectral fragmentation of quinoline alkaloids of significance in plants has been investigated using electrospray ionisation ion trap mass spectrometry (ESI-MSⁿ) with a view to characterisation of molecules of unknown structure isolated from these natural sources. This investigation has led to the generation of an appropriate database incorporating data from ESI-MSⁿ and also from gas liquid chromatography (GLC) and liquid chromatography (HPLC) for these low molecular mass quinolines. This has been put to practical application in the identification of quinoline alkaloids in a plant extract. Thus, an acid extraction of the leaves of Choisya ternata containing such tertiary alkaloids was analysed by liquid chromatography-electrospray ionisation mass spectrometry (HPLC-ESI-MS) and the resulting behaviour of the quinolines was compared with that of the quinoline alkaloids in the database.     

五味子中木脂素类成分的高效液相色谱-电喷雾质谱研究

采用液相色谱-电喷雾质谱联用(HPLC-ESI-MSⁿ)技术, 对北五味子与南五味子中木脂素类成分进行了系统研究. 通过HPLC-ESI-MS技术, 获得了相应化合物的保留时间、紫外光谱和分子量等信息, 利用电喷雾多级串联质谱技术(ESI-MSⁿ), 获得了相应化合物的结构信息. 研究结果表明, 北五味子与南五味子的主要木脂素成分除5个共有成分外其它成分差异较大, 并且其共有成分含量差别较大. 在此基础上, 建立了简便、快速的北五味子与南五味子药材分析鉴定的新方法.     

Direct identification of phenolic constituents in Boldo Folium (Peumus boldus Mol.) infusions by high-performance liquid chromatography with diode array detection and electrospray ionization tandem mass spectrometry

A very simple and direct method was developed for the qualitative analysis of polyphenols in boldo (Peumus boldus Mol., Monimiaceae) leaves infusions by high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization tandem mass spectrometry (HPLC-MSⁿ). The phenolic constituents identified in infusions of the crude drug Boldo Folium were mainly proanthocyanidins and flavonol glycosides. In the infusions, 41 compounds were detected in male and 43 compounds in female leaf samples, respectively. Nine quercetin glycosides, eight kaempferol derivatives, nine isorhamnetin glycosides, three phenolic acids, one caffeoylquinic acid glycoside and twenty one proanthocyanidins were identified by HPLC-DAD and ESI-MS for the first time in the crude drug. Isorhamnetin glucosyl-di-rhamnoside was the most abundant flavonol glycoside in the male boldo sample, whereas isorhamnetin di-glucosyl-di-rhamnoside was the main phenolic compound in female boldo leaves infusion. The results suggest that the medicinal properties reported for this popular infusion should be attributed not only to the presence of catechin and boldine but also to several phenolic compounds with known antioxidant activity. The HPLC fingerprint obtained can be useful in the authentication of the crude drug Boldo Folium as well as for qualitative analysis and differentiation of plant populations in the tree distribution range.     

Studies on the aconitine-type alkaloids in the roots of Aconitum Carmichaeli Debx. by HPLC/ESIMS/MS

Studies of aconitine-type alkaloids in the Chinese herb Aconitum Carmichaeli were performed by HPLC/ESIMS/MSⁿ and FTICR/ESIMS in positive ion mode. The characteristic fragmentation pathways in the MSⁿ spectra were summarized based on previously published research literature and further study. According to the fragmentation pathways of mass spectrometry, results from the analysis of standard compounds and reports from literature, 111 compounds were identified or deduced in a total of 117 found compounds in A. Carmichaeli. In the 11 monoester-diterpenoid alkaloids (MDA), 10 diesterditerpenoid alkaloids (DDA) and 81 lipo-alkaloids, the novel alkaloids including 1 MDA, 2 DDA and 48 lipo-alkaloids were detected. In addition, 1 DDA, 7 lipo-alkaloids and 2 alkaloids with small molecular weights that possess C19-norditerpenoid skeleton were reported in A. Carmichaeli for the first time.     

An investigation of bioactive phytochemicals in the leaves of Melicope vitiflora by electrospray ionisation ion trap mass spectrometry

The ethyl acetate extract of the leaves of Melicope vitiflora was separated by column chromatography and the resulting fractions tested for their bioactivity towards methicillin-resistant-Staphylococcus aureus (MRSA) and Micrococcus luteus (ML).The bioactive column chromatography fractions were further separated by preparative TLC and dereplication was carried out on them by first subjecting them to electrospray ionisation-ion trap mass spectrometry (ESI-MSⁿ). The resulting molecular masses, their fragmentation patterns in addition to the chemnet database (www.chemnetbase.com) were used to aid in the structural elucidation of some of the compounds by permitting comparison with known structures of natural origin. Some molecular masses and the corresponding fragmentations were found that did not correlate with any known compounds thus revealing potentially novel natural products that could be investigated on a larger scale and could ultimately find application as new drugs against MRSA and other multi drug resistant microorganisms. Structures are also proposed for known compounds that have not been previously reported for M. vitiflora.     

A study of the analytical behaviour of selected synthetic and naturally occurring coumarins using liquid chromatography, ion trap mass spectrometry, gas chromatography and polarography and the construction of an appropriate database for coumarin characterisation

The aim of this paper is to provide analytical chemical information on a range of naturally occurring and synthetic coumarins. This analytical chemical information on liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS), ion trap mass spectrometry (ESI-MSⁿ), gas chromatography-flame ionisation detection (GLC-FID) and polarographic behaviour is then incorporated into a database which is of use in identifying unknown coumarins isolated from natural sources. This paper is also concerned with understanding the effect of functional groups in coumarins on their analytical chemical behaviour using the above techniques.     

LC/ESI-MS n and 1H HR-MAS NMR analytical methods as useful taxonomical tools within the genus Cystoseira C. Agardh (Fucales; Phaeophyceae)

Species of the genus Cystoseira are particularly hard to discriminate, due to the complexity of their morphology, which can be influenced by their phenological state and ecological parameters. Our study emphasized on the relevance of two kinds of analytical tools, (1) LC/ESI-MSⁿ and (2) ¹H HR-MAS NMR, also called in vivo NMR, to identify Cystoseira specimens at the specific level and discuss their taxonomy. For these analyses, samples were collected at several locations in Brittany (France), where Cystoseira baccata, C. foeniculacea, C. humilis, C. nodicaulis and C. tamariscifolia were previously reported. To validate our chemical procedure, the sequence of the ITS2 has been obtained for each species to investigate their phylogenetic relationships at a molecular level. Our study highlighted the consistency of the two physico-chemical methods, compared to “classical” molecular approach, in studying taxonomy within the genus Cystoseira. Especially, LC/ESI-MSⁿ and phylogenetic analyses converged into the discrimination of two taxonomical groups among the 5 species. The occurrence of some specific signals in the ¹H HR-MAS NMR spectra and/or some characteristic chemical compounds during LC/ESI-MSⁿ analysis could be regarded as discriminating factors. LC/ESI-MSⁿ and ¹H HR-MAS NMR turned out to be two relevant and innovative techniques to discriminate taxonomically this complex genus.     

Characterisation of selected hypnotic drugs and their metabolites using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their determination by liquid chromatography-electrospray ionisation-ion trap mass spectrometry

The electrospray ionisation-ion trap mass spectrometry (ESI-MSⁿ) of selected hypnotic drugs, i.e. zopiclone, zolpidem, flunitrazepam and their metabolites have been investigated. Sequential product ion fragmentation experiments (MSⁿ) have been performed in order to elucidate the degradation pathways for the [M+H]⁺ ions and their predominant fragment ions. These MSⁿ experiments show certain characteristic fragmentations in that functional groups are generally cleaved from the ring systems as neutral molecules such as H₂O, CO, CO₂, NO₂, amines and HF. When an aromatic entity is present in a drug molecule together with a nitrogen-containing saturated ring structure as with zopiclone and its N-desmethyl metabolite fragmentation initially occurs at the latter ring with the former being resistant to fragmentation. The structures of fragment ions proposed for ESI-MSⁿ can be supported by electrospray ionisation-quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS).These molecules can be identified and determined in mixtures at low ng/ml concentrations by the application of liquid chromatography (LC)-ESI-MSⁿ which can be used for their analysis in saliva samples.This paper includes a tabulation of mass losses/signals at low m/z values for these hypnotic drugs and many others in recent publications which will be of value in the characterisation of drug metabolites of unknown structure and also natural product pharmaceuticals isolated from plants, etc.     

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.     

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.     

An MC3T3-E1 Cell Line Biomembrane Extraction and HPLC–ESI-MS n Method for Simultaneous Analysis of Potential Anti-Osteoporosis Components of Epimedium koreanum

Aerial parts of Epimedium koreanum Nakai have been used as Herba Epimedii in China. Its anti-osteoporosis effect has attracted much attention in recent years. In this study, a method involving osteoblastic cell (MC3T3-E1 cell line) extraction and high-performance liquid chromatography–electrospray ionisation–mass spectrometry (HPLC–ESI-MS ⁿ ) was developed for screening of potential anti-osteoporosis agents in E.?koreanum. Four compounds identified as epimedin?A, epimedin?B, epimedin?C and icariin were found to interact with MC3T3-E1 cells and possessed potent osteoblast-stimulating activity as evaluated by cell proliferation [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay] and differentiation [alkaline phosphatase (ALP) activity and Ca content] in?vitro. The results suggest that these four flavonoids are the anti-osteoporosis constituents of Herba Epimedii and that the method combining MC3T3-E1 cell extraction with HPLC–ESI-MS ⁿ is rapid and effective in screening anti-osteoporosis agents from traditional Chinese medicines.     

Chemical Characterization of Phenolic Compounds in Erigeron Injection by Rapid-Resolution LC Coupled with Multi-Stage and Quadrupole-TOF-MS

Rapid-resolution liquid chromatography coupled with electrospray ion trap multistage mass spectrometry (ESI-MS ⁿ ) and electrospray quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF) has been utilized to separate and characterize the phenolic components in Erigeron injections. After extensive separation on the column, each peaks’ potential structures were obtained based on their accurate mass supplied by ESI-Q-TOF and chemical database retrieval, and confirmed by ESI-MS ⁿ . A total of 44 compounds were identified or tentatively characterized and fifteen of them reported for the first time. In addition, the developed method was applied to monitor the elimination of phenolic compounds in rats after intravenous administration of 4 mL kg^?1 Erigeron injection. The caffeoylquinic acids and flavonoids of the Erigeron injection were quickly degraded and most of them reached the low limit of detection (SNR < 3) at 2 h post-injection.     

Medium-Pressure Liquid Chromatography Coupled to Electrospray Ionization Mass Spectrometry for Separation and On-Line Characterization of Flavonoids from Asparagus officinalis

A novel method for separation and on-line characterization of flavonoids from Asparagus officinalis by medium-pressure liquid chromatography coupled to electrospray ionization multi-stage mass spectrometry (MPLC-ESI-MSⁿ) was successfully established. The hyphenation between MPLC and ESI-MSⁿ was designed to keep the split ratio exactly in the range from 1:100 to 1:300. The separation procedure was guided by the chromatogram of ion current of MSⁿ and the structures of compounds were characterized by fragments information at the same time. Consequently, it was proved that MPLC coupled with ESI-MSⁿ was an effective method for separation of compounds from multi-component mixtures with high purity and desired amounts and simultaneous elucidation of chemical structures.

     

On-line identification of 4″-isovalerylspiramycin I in the genetic engineered strain of S. spiramyceticus F21 by liquid chromatography with electrospray ionization tandem mass spectrometry,ultraviolet absorbance detection and nuclear magnetic resonance spectrometry

LC-hyphenated techniques were applied to the on-line identification of isovalerylspiramycin I (isp I), a spiramycin-like macrolide in the crude extract of fermentation broth from a genetically engineered strain of S. spiramyceticus F21. In the structural characterization of the large molecular secondary metabolite of isp I, LC–DAD-UV–ESI-MSⁿ analysis played a crucial role, and stop-flow LC–¹H NMR measurement, with bitespiramycin used as reference, was a valuable complement approach. This rational approach proved to be an efficient means for the rapid and accurate structural determination of known microbial secondary metabolites, by which targeted isolation of component(s) of interest can be subsequently performed for further biological and pharmacological studies in drug development.     

Simultaneous quantification of three isoflavonoid glycosides in rabbit plasma after oral administration of Astragalus mongholicus extract by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A selective and sensitive liquid chromatography coupled with triple stage quadruple tandem mass spectrometry (HPLC/TSQ-MS/MS) was developed and validated for simultaneous quantification of calycosin-7-O-β-d-glycoside (CCSG), formononetin-7-O-β-d-glycoside (Ononin) and (6R,10R)-9,10-dimethoxypterocarpan-3-O-β-d-glycoside (DPG) in rabbit plasma. Plasma samples were extracted with solid-phase extraction (SPE), separated on an Inertsil ODS-3 column and detected by tandem mass spectrometry with electrospray ionization (ESI) interface in positive selective reaction monitoring (SRM) mode. 3,7,8-Trimethoxy-xanthone-1-O-primaverose was used as internal standard (IS) for quantitative measurement. For each analyte, one major product ion was chosen and used for screening of it. Calibration curves were generated over the range of 2-1000 ng mL⁻¹ with the correlation coefficients greater than 0.99 by using a weighted (1/χ) least squares linear regression. The method had the lower limit quantification of 0.15, 0.21 and 0.19 for CCSG, Ononin and DPG, respectively, with precision less than 20%. The intra- and inter-day precisions ranged from 2.48 to 6.38% and 4.81 to 11.78% (R.S.D.%), respectively. This assay is suitable for determining the above three trace glycosides in rabbit plasma simultaneously and thus investigating the pharmacokinetics of glycosides from Astragalus mongholicus extract in rabbits.