[Chemical constituents in different parts of Ixeris sonchifolia based on UPLC-LTQ-Orbitrap-MS~n].

The chemical constituents in stem leaf, root, and flower of Ixeris sonchifolia were identified by the ultra performance li-quid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS~n). The separation was performed on an Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 µm) with a mobile phase of water(containing 0.1% formic acid, A)-acetonitrile(B) with gradient elution. With electrospray ionization source, the data of 70% methanol extract from stem leaf, root and flower of I. sonchifolia were collected by high-resolution full-scan Fourier transform spectroscopy, data dependent acquisition, precursor ion scan, and selected ion monitoring in the negative and positive ion modes. The compounds were identified based on accurate molecular weight, retention time, fragment ions, comparison with reference standard, Clog P and references. A total of 131 compounds were identified from the 70% methanol extract of I. sonchifolia, including nucleosides, flavonoids, organic acids, terpenoids, and phenylpropanoids, and 119, 110, and 126 compounds were identified from the stem leaf, root and flower of I. sonchifolia, respectively. In addition, isorhamnetin, isorhamnetin-7-O-sambubioside and caffeylshikimic acid were discovered from I. sonchifolia for the first time. This study comprehensively analyzed and compared the chemical constituents in different parts of I. sonchifolia, which facilitated the discovery of effective substances and the development and application of medicinal material resources of I. sonchifolia.

Chemical constituents in different parts of Ixeris sonchifolia based on UPLC-LTQ-Orbitrap-MS~n / 中国中药杂志

The chemical constituents in stem leaf, root, and flower of Ixeris sonchifolia were identified by the ultra performance li-quid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS~n). The separation was performed on an Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) with a mobile phase of water(containing 0.1% formic acid, A)-acetonitrile(B) with gradient elution. With electrospray ionization source, the data of 70% methanol extract from stem leaf, root and flower of I. sonchifolia were collected by high-resolution full-scan Fourier transform spectroscopy, data dependent acquisition, precursor ion scan, and selected ion monitoring in the negative and positive ion modes. The compounds were identified based on accurate molecular weight, retention time, fragment ions, comparison with reference standard, Clog P and references. A total of 131 compounds were identified from the 70% methanol extract of I. sonchifolia, including nucleosides, flavonoids, organic acids, terpenoids, and phenylpropanoids, and 119, 110, and 126 compounds were identified from the stem leaf, root and flower of I. sonchifolia, respectively. In addition, isorhamnetin, isorhamnetin-7-O-sambubioside and caffeylshikimic acid were discovered from I. sonchifolia for the first time. This study comprehensively analyzed and compared the chemical constituents in different parts of I. sonchifolia, which facilitated the discovery of effective substances and the development and application of medicinal material resources of I. sonchifolia.

Mass spectral analysis of secondary metabolites from Zingiber montanum rhizome extract using UHPLC-HR-ESI-QTOF-MS/MS.

INTRODUCTION: Zingiber montanum (J.Koenig) Link ex A.Dietr. is a popular medicinal plant in Thailand. Its rhizomes have been used as an ingredient in various Thai traditional medicine formulas. While many reports have focused on the chemical constituents and biological activities of this plant, a comprehensive study on secondary metabolite profiling using tandem mass spectrometry has, to this point, never been documented. OBJECTIVE: To analyze the chemical constituents in Z. montanum rhizomes using ultra-high performance liquid chromatography coupled with ultra-high-resolution electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-HR-ESI-QTOF-MS/MS) analyses and to utilize the characteristic fragmentation patterns of these compounds to facilitate their identification. METHODOLOGY: UHPLC-HR-ESI-QTOF-MS/MS in positive ion mode was used for chemical identification of secondary metabolites from the ethanolic extract of the plant material. MS/MS data of some known reference compounds, together with detailed fragmentation pattern information of several compounds obtained from the crude extract, were used to elucidate their chemical structures. RESULTS: In this work, one benzaldehyde, ten phenylbutenoid monomers, six curcuminoids, and nine phenylbutenoid dimers were assigned based on their characteristic fragment ions. Among these compounds, 2-(3,4-dimethoxystyryl)oxirane was tentatively suggested as a potential new compound. Several characteristic fragment ions from these compounds were assigned and the relative ion abundance of these was also used to differentiate the chemical structures of compounds having the same molecular mass. CONCLUSIONS: The results will benefit future high-throughput screening of bioactive compounds and method development for the quality control of raw materials and herbal drugs derived from Z. montanum rhizome extracts.

Identification of Antiprotozoal Compounds from Buxus sempervirens L. by PLS-Prediction.

Various nor-triterpene alkaloids of Buxus (B.) sempervirens L. have shown remarkable in vitro activity against the causative agents of tropical malaria and East African sleeping sickness. To identify further antiprotozoal compounds of this plant, 20 different fractions of B. sempervirens L., exhibiting a wide range of in vitro bioactivity, were analyzed by UHPLC/+ESI-QqTOF-MS/MS. The analytical profiles were investigated by partial least squares regression (PLS) for correlations between the intensity of LC/MS signals, bioactivity and cytotoxicity. The resulting models highlighted several compounds as mainly responsible for the antiprotozoal activity and thus, worthwhile for subsequent isolation. These compounds were dereplicated based on their mass spectra in comparison with isolated compounds recently reported by us and with literature data. Moreover, an estimation of the cytotoxicity of the highlighted compounds was derived from an additional PLS model in order to identify plant constituents with strong selectivity. In conclusion, high levels of antitrypanosomal and antiplasmodial activity were predicted for eight and four compounds, respectively. These include three hitherto unknown constituents of B. sempervirens L., presumably new natural products.

Two New Alkaloids from Fuzi and Their Metabolites Study.

Former study suggests alkaloids from herbs of Aconitum genus plants possess excellent bioactivities, which exert great value for related deeper chemical constituent investigation. Herein, chemical isolation was performed and four alkaloids were isolated from Fuzi, of which two were new ones, and the other two were reported NMR data for the first time. Their chemical structures were identified by NMR data, high resolution MS, UV and IR analysis. Additionally, the MS fragmentation patterns were explored, formerly, that of hetisane alkaloid was rarely reported, and fragmentation mechanism of the diagnostic ion was proposed. Based on these fragment pathway, metabolites and metabolic pathways of four compounds were investigated in rat liver microsomes using UPLC-Q/TOF-MS, and dehydrogenation product was firstly found from metabolites of hetisane alkaloid.

[Study on fragmentation patterns of coumarins in Notopterygium inchum with ultrahigh performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry].

To demonstrate the fragmentation patterns of simple coumarins furanocourmarin(C_7-C_8), furanocourmarin(C_6-C_7) and dihydrofuran coumarin by mass spectrometry, with fraxin, scopoletin, isopsoralen, pimpinellin, isoimperatorin, notopterol and noda-kenin as study subjects, so as to provide a basis for rapid identification of compounds in different subtypes of coumarins. Ultrahigh performance liquid chromatography combined with quardrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was implemented in both positive and negative ion modes. Masslynx software was employed to provide the elemental constituents of each detected ion based on its accurate molecular weight. Chemdraw 2014 was used to cultivate mass number of each inferred structure. The fragment pattern of each compound was determined based on the structures inferred from all the relevant ions. And the patterns were drawn by Chemdraw 2014. The deviation between the calculated molecular weight of the inferred structure and the detected value of the ions was used to assess the correctness of the inferred structures in the fragmentation patterns. The results showed that with UPLC-Q-TOF, neutral loss of CO_2 and CO was reflected in lactone and furan skeletons from the courmarin structure. An even mass was attributed to the loss of an odd number of methyl radicals from compounds with a methoxy substituent. Furanocourmarin(C_7-C_8) produced a protonated molecular ion([M+H]~+), while the other courmarin subtypes produced either a sodium adduct of the molecular ion([M+Na]~+) or a sodium adduct of the molecular ion([M+Na]~+) with a protonated molecular ion([M+H]~+). The m/z 203.03 was a diagnostic ion for furanocourmarin(C_6-C_7), and the m/z 147.04 was supplementary evidence for furanocourmarin(C_6-C_7) identification. The characteristic ion of furanocourmarin(C_7-C_8) was m/z 131.05, while m/z 187.04 was the characteristic ion of dihydrofuran coumarin. The m/z 203.03 ion for furanocourmarin(C_7-C_8) was pretty weak. In negative ion mode, furanocourmarin(C_7-C_8) did not have any signals that were different from the other subtypes of courmarins. The fragmentation patterns in negative ion mode for the other subtypes of courmarins were similar to those in positive ion mode. Four types of fragmentation patterns were identified as forcourmarins from Notopterygium inchum. This study provides the basis for the rapid identification of courmarin subtypes by mass spectrometry.

Investigation of the Variability of Alkaloids in Buxus sempervirens L. Using Multivariate Data Analysis of LC/MS Profiles.

Buxus sempervirens L. is a common ornamental plant in southern and central Europe, and has been used ethopharmacologically against a wide variety of diseases due to it containing nor-triterpene alkaloids of the nor-cycloartane type. Recently, we demonstrated the interesting antiprotozoal potential of some of these compounds. To characterize the temporal variability in the alkaloid profile of two different varieties and their leaves and twigs, 30 different extracts of B. sempervirens were evaluated by Ultra High Performance Liquid Chromatography/positive Mode-Electrospray Ionization Quadrupole Time-of-Flight-Tandem Mass Spectrometry (UHPLC/+ESI-QqTOF-MS/MS). The analytical profiles were thoroughly investigated by various methods of multivariate data analysis (MVDA). A principal component analysis (PCA) model elucidates the seasonal variation in the phytochemical composition of B. sempervirens var. arborescens and suffruticosa along with differences between the varieties. Analysis of a volcano plot illustrated the differences between the two organs, the leaf and twig. Eighteen compounds were highlighted by the models as constituents of the plant characteristic for a season, variety or organ. These compounds were dereplicated based on their chromatographic and +ESI-QqTOF-MS and -MS/MS data. In addition, mass spectral fragmentation pathways for already known alkaloids as well as new natural products could be postulated for the first time. In conclusion, the MVDA models give detailed information on the temporal variability in the alkaloid profile of two different varieties and their organs (leaf vs. twig) of B. sempervirens. Thus, the results of this study allow, e.g., the identification of characteristic compounds for the different varieties, plant organs, seasons, and the optimal harvesting time for the isolation of particular Buxus-alkaloids of interest for subsequent studies.

Study on fragmentation patterns of coumarins in Notopterygium inchum with ultrahigh performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry / 中国中药杂志

To demonstrate the fragmentation patterns of simple coumarins furanocourmarin(C_7-C_8), furanocourmarin(C_6-C_7) and dihydrofuran coumarin by mass spectrometry, with fraxin, scopoletin, isopsoralen, pimpinellin, isoimperatorin, notopterol and noda-kenin as study subjects, so as to provide a basis for rapid identification of compounds in different subtypes of coumarins. Ultrahigh performance liquid chromatography combined with quardrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was implemented in both positive and negative ion modes. Masslynx software was employed to provide the elemental constituents of each detected ion based on its accurate molecular weight. Chemdraw 2014 was used to cultivate mass number of each inferred structure. The fragment pattern of each compound was determined based on the structures inferred from all the relevant ions. And the patterns were drawn by Chemdraw 2014. The deviation between the calculated molecular weight of the inferred structure and the detected value of the ions was used to assess the correctness of the inferred structures in the fragmentation patterns. The results showed that with UPLC-Q-TOF, neutral loss of CO_2 and CO was reflected in lactone and furan skeletons from the courmarin structure. An even mass was attributed to the loss of an odd number of methyl radicals from compounds with a methoxy substituent. Furanocourmarin(C_7-C_8) produced a protonated molecular ion([M+H]~+), while the other courmarin subtypes produced either a sodium adduct of the molecular ion([M+Na]~+) or a sodium adduct of the molecular ion([M+Na]~+) with a protonated molecular ion([M+H]~+). The m/z 203.03 was a diagnostic ion for furanocourmarin(C_6-C_7), and the m/z 147.04 was supplementary evidence for furanocourmarin(C_6-C_7) identification. The characteristic ion of furanocourmarin(C_7-C_8) was m/z 131.05, while m/z 187.04 was the characteristic ion of dihydrofuran coumarin. The m/z 203.03 ion for furanocourmarin(C_7-C_8) was pretty weak. In negative ion mode, furanocourmarin(C_7-C_8) did not have any signals that were different from the other subtypes of courmarins. The fragmentation patterns in negative ion mode for the other subtypes of courmarins were similar to those in positive ion mode. Four types of fragmentation patterns were identified as forcourmarins from Notopterygium inchum. This study provides the basis for the rapid identification of courmarin subtypes by mass spectrometry.

Diagnostic fragmentation-assisted mass spectral networking coupled with in silico dereplication for deep annotation of steroidal alkaloids in medicinal Fritillariae Bulbus.

Fully understanding the chemicals in an herbal medicine remains a challenging task. Molecular networking (MN) allows to organize tandem mass spectrometry (MS/MS) data in complex samples by mass spectral similarity, which yet suffers from low coverage and accuracy of compound annotation due to the size limitation of available databases and differentiation obstacle of similar chemical scaffolds. In this work, an enhanced MN-based strategy named diagnostic fragmentation-assisted molecular networking coupled with in silico dereplication (DFMN-ISD) was introduced to overcome these obstacles: the rule-based fragmentation patterns provide insights into similar chemical scaffolds, the generated in silico candidates based on metabolic reactions expand the available natural product databases, and the in silico annotation method facilitates the further dereplication of candidates by computing their fragmentation trees. As a case, this approach was applied to globally profile the steroidal alkaloids in Fritillariae bulbus, a commonly used antitussive and expectorant herbal medicine. Consequently, a total of 325 steroidal alkaloids were discovered, including 106 cis-D/E-cevanines, 142 trans-D/E-cevanines, 29 jervines, 23 veratramines, and 25 verazines. And 10 of them were confirmed by available reference standards. Approximately 70% of the putative steroidal alkaloids have never been reported in previous publications, demonstrating the benefit of DFMN-ISD approach for the comprehensive characterization of chemicals in a complex plant organism.

Systematic characterization of the chemical constituents in vitro and prototypes in vivo of Dingkun Dan using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with the UNIFI™ software.

Dingkun Dan (DKD), a famous traditional Chinese medicine, has been widely used in the treatment of irregular menstruation, leucorrhea abnormality, and postpartum gynecological diseases since Qing dynasty (1739). It comprises 30 flavors of Chinese medicinal materials, which results in its complex chemical composition. In this study, an integrative method was developed to rapidly characterize the chemical components of DKD using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with the UNIFI™ software. A total of 234 compounds, including 47 triterpenoid saponins, 55 flavonoids, and 38 alkaloids, were identified. Of them, 170 compounds were characterized initially and 61 compounds were identified unambiguously using reference standards. Under the same analysis conditions, 43 prototypical components, which were tentatively assigned as triterpenoid saponins, flavonoids, alkaloids, terpenoids, phenylpropanoids, and others, were absorbed in rat by serum pharmacochemistry analysis. DKD exhibited diverse pharmacological activities through the combined effect of these components. This study was the first systematic study of chemical components in vitro originating from 30 medicinal materials and prototypes in vivo of DKD, which could provide scientific evidence for explaining its therapeutic effect.

Comparison of the Polyphenolic Profile of Medicago sativa L. and Trifolium pratense L. Sprouts in Different Germination Stages Using the UHPLC-Q Exactive Hybrid Quadrupole Orbitrap High-Resolution Mass Spectrometry.

Identification and quantification of polyphenols in plant material are of great interest since they make a significant contribution to its total bioactivity. In the present study, an UPLC-Orbitrap-MS/MS approach using the variable data acquisition mode (vDIA) was developed and applied for rapid separation, identification, and quantification of the main polyphenolic compounds in Medicago sativa L. and Trifolium pratense L. sprouts in different germination stages. Based on accurate MS data and fragment ions identification strategy, a total of 29 compounds were identified by comparing their accurate masses, fragment ions, retention times, and literatures. Additionally, a number of 30 compounds were quantified by comparing to the reference standards. Data were statistically analysed. For both plant species, the sprouts of the third germination day are valuable sources of bioactive compounds and could be used in phytotherapy and nutrition. Although Trifolium pratense L. (Red Clover) is considered to be a reference for natural remedies in relieving menopause disorders, alfalfa also showed a high level of biological active compounds with estrogenic activity.

Electron Transfer Induced Decomposition in Potassium-Nitroimidazoles Collisions: An Experimental and Theoretical Work.

Electron transfer induced decomposition mechanism of nitroimidazole and a selection of analogue molecules in collisions with neutral potassium (K) atoms from 10 to 1000 eV have been thoroughly investigated. In this laboratory collision regime, the formation of negative ions was time-of-flight mass analyzed and the fragmentation patterns and branching ratios have been obtained. The most abundant anions have been assigned to the parent molecule and the nitrogen oxide anion (NO2-) and the electron transfer mechanisms are comprehensively discussed. This work focuses on the analysis of all fragment anions produced and it is complementary of our recent work on selective hydrogen loss from the transient negative ions produced in these collisions. Ab initio theoretical calculations were performed for 4-nitroimidazole (4NI), 2-nitroimidazole (2NI), 1-methyl-4- (Me4NI) and 1-methyl-5-nitroimidazole (Me5NI), and imidazole (IMI) in the presence of a potassium atom and provided a strong basis for the assignment of the lowest unoccupied molecular orbitals accessed in the collision process.

Phytochemical Profiling of Coryphantha macromeris (Cactaceae) Growing in Greenhouse Conditions Using Ultra-High-Performance Liquid Chromatography⁻Tandem Mass Spectrometry.

Chromatographic separation combined with mass spectrometry is a powerful tool for the characterization of plant metabolites because of its high sensitivity and selectivity. In this work, the phytochemical profile of aerial and radicular parts of Coryphantha macromeris (Engelm.) Britton & Rose growing under greenhouse conditions was qualitatively investigated for the first time by means of modern ultra-high-performance liquid chromatography⁻tandem mass spectrometry (UHPLC-PDA-HESI-Orbitrap-MS/MS). The UHPLC-PDA-HESI-Orbitrap-MS/MS analysis indicated a high complexity in phenolic metabolites. In our investigation, 69 compounds were detected and 60 of them were identified. Among detected compounds, several phenolic acids, phenolic glycosides, and organic acids were found. Within this diversity, 26 metabolites were exclusively detected in the aerial part, and 19 in the roots. Twenty-four metabolites occurred in both plant parts. According to the relative abundance of peaks in the chromatogram, ferulic and piscidic acids and their derivatives may correspond to one of the main phenolic compounds of C. macromeris. Our results contribute to the phytochemical knowledge regarding C. macromeris and its potential applications in the pharmaceutical and cosmetic industries. Besides, some metabolites and their fragmentation patterns are reported here for the first time for cacti species.

Liquid Chromatography Coupled with Linear Ion Trap Hybrid OrbitrapMass Spectrometry for Determination of Alkaloids in Sinomeniumacutum.

The characterization of alkaloids is challenging because of the diversity of structures and the complicated fragmentation of collision induced structural dissociation in mass spectrometry. In this study, we analyzed the alkaloids in Sinomenium acutum (Thunb.) Rehderet Wil by high resolution mass spectrometry. Chromatographic separation was achieved on a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 µm) column with a mobile phase consisting of acetonitrile and water (0.1% formic acid) under gradient elution. A total of 52 alkaloids were well separated and 45 of them were structurally characterized, including morphinans, aporphines, benzylisoquinolines, and protoberberines. Specially, mass spectrometric study of the morphinan alkaloids were explicitly investigated. Electrostatic potential plot from simulation was calculated for determination of protonation sites. Further fragmentation analysis suggested that the C3H7N, CH4O, and H2O elimination was displayed in MS² spectrum. These fragmentation pathways are universal for morphinan alkaloids having methoxy substituted cyclohexenone or cyclohexadienone moieties. Additionally, for nitrogen oxides, an ion-neutral complex intermediate is involved in the fragmentation process, generating additional oxygenated ions. All these results provided the universal rules of fragmentation used for detection of alkaloids, and will be expected to be highly useful for comprehensive study of multi-components in the herbal medicine analysis.

Characterization and identification of isoflavonoid glycosides in the root of Spiny restharrow (Ononis spinosa L.) by HPLC-QTOF-MS, HPLC-MS/MS and NMR.

Restharrow root has been used in traditional medicine for thousands of years; however, the active ingredients responsible for the diuretic effect are still unknown. Previous studies have proved that the root extract contains isoflavonoids, however only few derivatives were identified, mostly relying on retention times or UV data. The aim of our work was to perform a detailed structural characterization of the complete isoflavonoid profile in the aqueous-methanolic extract of Ononis spinosa root by high-performance liquid chromatography coupled with electrospray ionization accurate-mass quadrupole time-of-flight and tandem mass spectrometry in positive ionization mode (HPLC-ESI-QTOF-MS, HPLC-ESI-MS/MS) and nuclear magnetic resonance spectroscopy (NMR). On the basis of the accurate masses and fragmentation patterns isoflavones (formononetin, calycosin and pseudobaptigenin) and pterocarpans (maackiain and medicarpin) were identified. Two further dihydroisoflavone aglycones, namely onogenin and sativanone and a unique glucoside were isolated and their structures were elucidated by NMR experiments. Calycosin, onogenin and sativanone were detected in this plant for the first time. In contrast to previous works, the presence of biochanin A could not be confirmed, however its regioisomer calycosin and its derivatives were identified. Similarly, neither tectorigenin derivatives could be detected, however the isobar compound sativanone and its various glucosides were elucidated. The presence of genistein and daidzein could not be confirmed in the extract. Fragmentation pathways for onogenin and sativanone are presented. In the aqueous-methanolic extract 9 glucosides, 6 minor and 8 major glucoside malonates, 4 glucoside acetates and 7 aglycones were found. In total, 34 compounds were successfully identified.

[Rapid identification of chemical components in Congrong Zonggan capsule by UPLC-Q-TOF-MS/MS].

This study was aimed to qualitatively analyze the chemical components in Congrong Zonggan capsule by using ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry method (UPLC-Q-TOF-MS/MS). An Agilent SB-C18 Rapid Resolution HD (3.0 mm×100 mm,1.8 µm) was used with acetonitrile (A) - 0.1% formic acid solution (B) as the mobile phase for gradient elution. The flow rate was 0.2 mL•min⁻¹; the detection wavelength was set at 330 nm and the column temperature was maintained at 30 ℃. Electrospray ion (ESI) source was applied for the qualitative analysis under the negative ion mode. Finally, based on comparison with standard samples, database matching analysis and reviewing relevant literature, 41 compounds were identified from Congrong Zonggan capsule. This method could be used to rapidly detect the chemical components in Congrong Zonggan capsule, providing reference for the quality control of Congrong Zonggan capsule and laying a foundation for the further study on active components mechanism.