Meliacarpinin-Type Limonoids from the Bark of Melia toosendan.

Three new meliacarpinin-type limonoids, toosendanes A⁻C (1⁻3), along with three, known meliacarpinins (4⁻6) were isolated from the bark of Melia toosendan. Their structures, along with their absolute configurations, were elucidated, based on detailed analyses. These included HRESIMS and 1D/2D-NMR, modified Mosher's method, and electronic circular dichroism (ECD). Limonoids 2 and 3 showed moderate inhibitory activity on LPS-activated, RAW 264.7 macrophages.

One-step targeted accumulation and detection of camptothecin analogues from fruits of Camptotheca acuminata Decne using bilayer solid-phase extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry.

Camptothecins, a kind of monoterpene-quinoline alkaloids from Camptotheca acuminata Decne, have long attracted much attention worldwide as an anti-cancer drug. However, there is still a lack of effective methods for the accumulation and discovery of camptothecin analogues from botanic resources for camptothecin-based drug research. This work develops a one-step method for the targeted accumulation, quick detection, and identification of camptothecin analogues from C. acuminata fruit using bilayer solid-phase extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry (bilayer-SPE-UHPLC-Q-TOF-MS/MS). The bilayer-SPE cartridge, with polyamide (PA) as the upper layer and octadecyl silane (ODS) as the lower layer, was designed for the removal of flavonoid and ellagic acid impurities and the enrichment of camptothecins for further MS analysis. Subsequently, the mass spectrometry fragmentations, especially multistage retro-Diels-Alder cleavage, were summarized based on the MS/MS data of 10 reference camptothecins. The UHPLC-Q-TOF-MS/MS conditions were optimized, and the MS/MS data of the potential camptothecin analogues in the bilayer-SPE enriched fractions were analyzed. A total of 30 camptothecin analogues, including 15 new compounds, were identified from the fruit according the fragmentation pathways of the reference standards. The proposed structure of peak 20 was confirmed using its NMR data through rapid enrichment and purification. Overall, the bilayer-SPE enrichment and reliable mass spectrometry fragmentation in our work could provide an effective and simple method for the exploration of the biosynthesis pathway and metabolomics of camptothecin analogues.

Hydrogen/deuterium exchange, a unique and effective method for MS fragmentation behavior elucidation of ginkgolides and its application to systematic research in Ginkgo biloba.

Ginkgolides, the main active constituents of Ginkgo biloba, possess significant selectively inhibition on platelet-activating factor and pancreatic lipase and attract wide attention in pharmacological research area. In our study, an effective hydrogen/deuterium (H/D) exchange method was developed by exchanging the α-Hs of lactone groups in ginkgolides with Ds, which was very useful for the elucidation of the fragmentation patterns of ginkgolides in Quadrupole Time-of-flight Mass Spectrometry (Q-TOF-MS), especially in accurately distinguishing the type and position of substituent in framework of ginkgolides. Then, a systematic research strategy for qualitative and quantitative analysis of ginkgolides, based on H/D exchange, tandem solid-phase extraction and LC-Q-TOF-MS, was developed, which was successfully applied in each medicinal part of G. biloba, which indicated that ginkgolide B was the most abundant ginkgolide in the seeds of G. biloba (60.6µg/g). This research was the successful application of H/D exchange in natural products, and proved that H/D exchange is a potential method for analysis research of complex TCMs active constituents.

Tandem solid-phase extraction followed by HPLC-ESI/QTOF/MS/MS for rapid screening and structural identification of trace diterpenoids in flowers of Rhododendron molle.

INTRODUCTION: 'Naoyanghua', composed of the flowers of Rhododendron molle G. Don, is a traditional Chinese medicine that is widely known for its toxicity. Grayanane-type diterpenoids are the main active ingredients in R. molle, as well as possibly their toxicity: they are, however, difficult to isolate and analyse using common chromatographic methods, due to their small amounts and absence of conjugated groups, such as phenyl and α, ß-unsaturated ketone. OBJECTIVE: To establish a highly sensitive, selective and reliable method for the qualitative evaluation of trace diterpenoids in the flowers of R. molle by using tandem solid-phase extraction followed by high-performance liquid chromatography with electrospray ionisation quadrupole-time-of-flight mass spectrometry (HPLC-ESI/QTOF/MS/MS). METHODS: Tandem solid phase extraction (SPE) was undertaken using a polyamide cartridge and a C18E cartridge in succession to enrich the trace diterpenoids. HPLC-ESI/QTOF/MS/MS was used to determine the fragmentation patterns of diterpenoids and to tentatively characterise their fragmentation pathways. RESULTS: HPLC-ESI/QTOF/MS/MS detected a total of 14 diterpenoids, eight of which were identified by comparison with literature sources and six based on fragmentation analysis. Among the latter six, rhodojaponin VI-3-glucoside was tentatively identified as a new diterpenoid glycoside and rhodojaponin VII, rhodojaponin IV and rhodojaponin I were reported from R. molle for the first time. CONCLUSION: By qualitative research of diterpenoids in this plant by HPLC-ESI/QTOF/MS/MS, a reliable methodology for the analysis of these active constituents of R. molle was established for the first time.

Characterisation of homoflavonoids from three Ophioglossum species using liquid chromatography with diode array detection and electrospray ionisation tandem mass spectrometry.

INTRODUCTION: Homoflavonoids, characterised by one more carbon atom directly added to C6 -C3 -C6 backbone of flavonoids, are rich in the species of genus Ophioglossum. Up to now we have little knowledge about their MS fragmentation patterns. It is therefore necessary to investigate their MS fragmentation pathways so as to distinguish them from other types of flavonoids. OBJECTIVE: To develop a rapid method for identifying homoflavonoids from Ophioglossum based on their characteristic MS fragmentation. METHODS: Mass spectrometry fragmentation pathways and qualitative analysis of homoflavonoids in three ferns of Ophioglosssum were investigated by using high-performance liquid chromatography coupled with diode-array detection and electrospray ionisation tandem mass spectrometry (HPLC-DAD-ESI/MS(n) ). RESULTS: The analyses of the MS(n) spectra of the homoflavonoids allowed us to classify them into two types according to their fragmentation characteristics. The type I homoflavonoids, with an attached additional carbon atom to the C-3 position of the C-ring, presented the initial competing loss of H2 O and CH2 O from their aglycone ions, compared to the initial removal of H2 O or CO in the case of the type II homoflavonoids, which bear the additional carbon atom at the C-2' site of the B-ring and forming ring D. The above characteristic fragmentations of homoflavonoids were quite different from those of other flavonoids, and were successfully applied to identify homoflavonoids in the crude extracts of three Ophioglossum species. CONCLUSION: The HPLC-DAD-ESI/MS(n) method obtained in the present study provided a powerful tool for identifying homoflavonoids from ferns in the genus Ophioglossum.

New features on the fragmentation patterns of homoisoflavonoids in Ophiopogon japonicus by high-performance liquid chromatography/diode-array detection/electrospray ionization with multi-stage tandem mass spectrometry.

Homoisoflavonoids, a special class of flavonoids, are mainly distributed in the Liliaceae family and have various biological activities. Previously, very little research has been reported on the gas-phase fragmentation patterns of homoisoflavonoids by electrospray ionization mass spectrometry. In this paper, we report the use of high-performance liquid chromatography with a diode-array detector (HPLC-DAD) and electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) to study the fragmentation behavior of 11 homoisoflavonoid standards and to analyze homoisoflavonoids in Ophiopogon japonicus. In total, 28 homoisoflavonoids (including seven novel constituents) were characterized. The deprotonated [M--H](-) molecules of the homoisoflavonoids containing a saturated C2--C3 bond afforded the A or B product ion (base peak) according to whether the B-ring was substituted with a hydroxyl group. For the homoisoflavonoids containing a C-2-C-3 double bond, the product ions (A or C ion) were created from the precursor [M-H](-) ion as the base peak when the B-ring was substituted with a hydroxyl group. The homoisoflavonoids carrying a formyl group in the A-ring readily eliminated one molecule of CO to form the product ion [M + H-CO](-) (base peak) irrespective whether the C-2-C-3 bond was saturated or not. This product ion afforded the [M-H-CO-B-ring--CH(2) + H](-) ion by cleavage of the C3-C9 bond. This latter product ion always appeared in tandem mass (MS/MS) spectra of type I homoisoflavonoids. The common features of flavonoids observed during the gas-phase fragmentation mechanisms were the loss of the following groups: 15 Da (CH(3)), 18 Da (H(2)O), 28 Da (CO), 44 Da (CO(2)) and 46 Da (CH(2)O(2)). A retro-Diels-Alder (RDA)-like cleavage was also observed for the homoisoflavonoids. The different gas-phase fragmentation routes were characterized for the deprotonated molecules obtained from the various homoisoflavonoids and collision-induced dissociation (CID) fragmentation differences were noted for the different locations of the various substituents. In conclusion, we can say that this study allowed us to structurally elucidate and identify homoisoflavonoids distributed in related plants and their complex prescriptions.

Qualitative and quantitative analysis of steroidal saponins in crude extracts from Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis by high performance liquid chromatography coupled with mass spectrometry.

High performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC-ESI-MS(n)) and triple quadrupole mass spectrometric detection (HPLC-ESI-MS/MS), respectively, had been employed for the simultaneous identification and quantification of steroidal saponins in the rhizomes of Parispolyphylla var. yunnanensis and P. polyphylla var. chinensis, which are the qualified plants of "Chonglou" in Chinese. The HPLC experiments were performed by means of a reversed-phase C-18 column and a binary mobile phase system consisting of 0.1% aqueous formic acid and acetonitrile under gradient elution conditions. The characteristic fragmentation patterns of diosgenin- and pennogenin-type steroidal saponins were investigated using ESI-MS(n) in negative ion mode. The MS(n) data of the [M-H](-) ions provided structural information on the sugar sequence of the oligosaccharide chains and the aglycones of steroidal saponins. As a result, ten and seven saponins were determined in P. polyphylla var. yunnanensis and P. polyphylla var. chinensis, respectively, including four unknown compounds. One unknown compound was tentatively identified as diosgenin-3-O-alpha-L-rhamnopyranosyl(1-->4) [alpha-L-rhamnopyranosyl(1-->2)]-beta-D-glucopyranoside and the aglycones of the other three new compounds were reported from Chonglou for the first time. The developed HPLC-ESI-MS/MS method was validated and found to be satisfactorily linear, selective and robust. The limits of detection (LODs) and quantitation (LOQs) ranged, respectively, from 0.5 to 10 ng/mL and 2 to 34 ng/mL depending on six various compounds. The intra- and inter-day precisions of the method were evaluated and were less than 5.0%. Recoveries ranged from 92% to 104% for all compounds. The established quality evaluation method was successfully used for simultaneous quantification of six predominant steroidal saponins in the rhizomes of these two Paris species.

Rapid identification of two species of Peucedanum by high-performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry.

The fragmentation behaviors of the angular- and linear-type coumarins from Peucedanum praeruptorum Dunn and P. decursivum (Miq.) Maxim were simultaneously investigated by HPLC-DAD/ESI-MS(n). For more structural identification, the fragment ions were analyzed and some possible fragmentation pathways were proposed. Different positions and numbers of the substituent also led to different fragment behaviors. Two types of coumarins from P. praeruptorum and P. decursivum were structurally elucidated by these techniques. In addition, UV spectra were applied to support the MS analysis. This is the first time that the two types of coumarins from herbal extracts have been differentiated by HPLC-DAD/ESI-MS(n). The method further illustrated the importance of the ESI-MS(n) technique in the identification of different types of coumarins and was applied for the rapid differentiation of the two herbs.

An application of high-speed counter-current chromatography coupled with electrospray ionization mass spectrometry for separation and online identification of coumarins from Peucedanum praeruptorum Dunn.

A new and systematic application for separation and online identification of coumarins from Peucedanum praeruptorum Dunn by preparative high-speed counter-current chromatography coupled with electrospray ionization multi-stage mass spectrometry (prep-HSCCC/ESI-MS(n)) was established. The procedure of separation was guided by the chromatogram of ion current. The structures of acquisitions were deduced by MS information. The hyphenation between prep-HSCCC/ESI-MS(n) was designed to keep the split ratio from 1:20 to 1:200 exactly. Seven compounds were obtained and two new compounds were detected. It was proved that prep-HSCCC/ESI-MS(n) was an effective method for sensitive detection, rapid identification and separation of natural products.

Characterization of spirostanol saponins in Solanum torvum by high-performance liquid chromatography/evaporative light scattering detector/electrospray ionization with multi-stage tandem mass spectrometry.

High-performance liquid chromatography with an evaporative light scattering detector and electrospray ionization multistage tandem mass spectrometry (HPLC/ELSD/ESI-MS(n)) was used to identify spirostanol saponins in a saponin extract of Solanum torvum. The fragmentation behavior of saponins was studied using ESI-MS(1-3) in positive ion mode. The MS(n) spectra of the [M+H](+) ions provide structural information including aglycone type and the nature and sequence of sugars. The use of ELSD allowed the profiling of the nonchromophore-containing saponins in this plant. The MS analysis established in this study with known saponins was successfully applied to tentatively identify two new siprostanol glycosides, neosolaspigenin 6-O-beta-D-quinovopyranoside and solagenin 6-O-[beta-D-xylopyranosyl-(1 --> 3)-O-beta-D-quinovopyranoside].

[HPLC-MS3 analysis of chemical constituents in Epimedium brevicornum].

OBJECTIVE: To investigate the chemical constituents of effective part of Epimedium brevicornum. METHOD: The sample was extracted with ethanol and purified by macroporous resin. The structures were identified by HPLC-MS3 experiments. RESULT: Nine compounds were identified from the effective part of E. brevicornum. CONCLUSION: The method is simple and rapid for the identification of the flavonoids from E. brevicornum.

[Determination of linderane in root tuber of Lindera aggregata by HPLC].

OBJECTIVE: To provide scientific basis for quality control of Lindera aggregata. METHOD: HPLC analytical method was established using a Lichrospher C18 column and acetonitrile-water (56:44) as the mobile phase, detected at 235 nm. RESULT: The linear range of linderane is between 0.0642 - 0.5774 microg, the average recovery was 98.4%, RSD1.7% (n = 9). CONCLUSION: Contents of linderane in commercially available and collected samples were from 0.028% to 0.123% and from 0.056% to 0.222% respectively.

[Detection of hepatotoxic pyrrolizidine alkaloids in Ligularia Cass. with LC/MSn].

AIM: To detect the hepatotoxic pyrrolizidine alkaloids (HPA) in the genus Ligularia Cass.. METHODS: The alkaloid extracts of Ligularia plant materials were detected and analyzed by the method of combination of TLC, and LC/MSn. RESULTS: Among 22 species of Ligularia Cass., HPA were detected in 18 species with LC/MSn, and no HPA was detected in the remaining 4 species. CONCLUSION: HPA was first detected with LC/MSn in L. tongelensis and other 15 species of Ligularia Cass.; HPA from these plants should be isolated, separated and identified and it is necessary to study the activities and toxicities of the HPA. The types and kinds of HPA from different species and sources are different, they should be detected separately.