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.

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.

[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.