Liquid chromatography with ultraviolet absorbance-mass spectrometric detection and with nuclear magnetic resonance spectroscopy: a powerful combination for the on-line structural investigation of plant metabolites

In order to discover new bioactive compounds from plant sources which could become new leads or new drugs, extracts should be submitted at the same time to chemical screening and to various biological or pharmacological targets. Metabolite profiling using hyphenated techniques such as LC/UV, LC/MS and more recently LC/NMR, quickly provides plenty of structural information, leading to a partial or a complete on-line de novo structure determination of the natural products of interest. As a complement to this approach, bioassays performed after LC/microfractionation of the extracts allow efficient localisation of the bioactive LC-peaks in the chromatograms. The combination of metabolite profiling and LC/bioassays provides the possibility of distinguishing between already known bioactive compounds (dereplication) and new molecules directly in crude plant extracts. Thus, the tedious isolation of compounds of low interest can be avoided and targeted isolation of new bioactive products or constituents presenting novel or unusual spectroscopic features can be undertaken. Several examples of rapid localisation of bioactive compounds, based on post-chromatographic bioautographic testing of LC/NMR microfractions and subsequent on-line identification will be illustrated. Application of hyphenated techniques for the efficient characterisation of labile constituents or constituents difficult to separate at the preparative scale will also be mentioned. The possibilities and limitations of LC/UV/NMR/MS and LC/bioassay as well as future development expected in this field will be discussed.     

Phytochemistry in the microgram domain - a LC-NMR perspective

Plants represent an extraordinary reservoir of novel molecules and there is currently a resurgence of interest in the vegetable kingdom as a possible source of new lead compounds for introduction into therapeutical screening programs. In order to discover potential new bioactive natural products, the dereplication of crude plant extracts performed prior to isolation work is of crucial importance for avoiding the isolation of a known constituent. In this respect, chemical screening strategies have been developed using hyphenated techniques (LC/UV-DAD, LC-MS and LC-NMR). In our laboratory, these techniques have been fully integrated into the isolation process and are used for the chemical screening of crude plant extracts in complement with on-line or at-line bioassays. LC-UV-MS is used as a first dereplication step in combination with UV and MS databases, while LC-NMR is performed in a second step for de novo on-line structure determination. This approach enables the partial or the complete on-line identification of natural products in complex matrices such as crude plant extracts. These methods also give a unique possibility to study unstable compounds, which rapidly degrade or which are not separable at a preparative level.In the multi-hyphenated approach used (hypernation), LC-NMR plays a key role since it provides the most detailed structural information. The relatively low sensitivity of this technique, however, requires that strategies for high loading of plant extracts are developed and compromises for solvent selection have to be made. For more demanding experiments, at-line strategies based on the microfractionation of the LC-peak of interest and recording of spectra in fully deuterated solvents in microflow probes represent a promising alternative.     

Hyphenated chromatographic techniques for the rapid screening and identification of antioxidants in methanolic extracts of pharmaceutically used plants

Phytochemical analysis is an important scientific research area, which normally relies on a number of rather laborious and time-consuming techniques for compound identification. Isolation of the ingredients of plant extracts in adequate quantities for spectral and biological analysis was the basis of this research. In this paper the possibility of on-line rapid screening of antioxidant components in methanolic plant extracts and their subsequent identification is reported. Based exclusively on hyphenated chromatographic techniques the methanolic extracts of Tilia europea, Urtica dioica, Lonicera periclymenum and Hypericum perforatum are initially screened for their antioxidant components via an on-line DPPH and ABTS radical scavenging technique. Structural elucidation of the active analytes is achieved by means of LC-MS and LC-UV-SPE-NMR. After the determination of the appropriate LC gradient, a minimal number of chromatographic runs with these hyphenated techniques are adequate for the acquisition of the necessary data, leading to the identification of the targeted compounds. Based on their UV, NMR and MS spectra, the antioxidant compounds identified in the extracts under study were found to be either flavonoid glycosides or mono- and dicaffeoylquinic acids. Although the aim of the study was to show the great potential of the LC-UV-NMR-DPPH/ABTS approach for the rapid screening and identification of plant constituents, the results produced in the course of this study also have some merit by themselves. Some of the compounds detected are reported for the first time in the specific plant extracts.     

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).     

Separation and identification of multiple constituents in Xiao Chai Hu Decoction (Sho‐saiko‐to) by bioactivity‐guided fractionation combined with LC‐ESI‐QTOFMS/MS

Xiao Chai Hu Decoction (XCHD), named Sho‐saiko‐to in Japanese, is a well‐known traditional Chinese medicine formula used in Asia. However, the characterization methods used in the past have lacked sensitivity and the nature of the active constituents of XCHD remains unclear. This study was carried out to establish the hyphenated method of bioactivity‐guided fractionation and liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (LC‐ESI‐QTOFMS/MS) in order to identify the major bioactive constituents of XCHD. D101 macroporous resin was used to separate and enrich the material base into four fractions, XCHD‐1, XCHD‐2, XCHD‐3 and XCHD‐4. Each fraction was then evaluated for its antidepressant effect using depression‐related parameters. An LC‐ESI‐QTOFMS/MS method in both positive and negative ion mode was also applied for separation and identification of the biological active fractions of XCHD. As a result, 79 compounds including polysaccharides, flavonoids, saikosaponins, ginsenosides, licoricesaponins and gingerols were detected, 69 of them were identified or tentatively characterized. Based on our preliminary characterization investigations, polysaccharides, gingerols and flavonoids in XCHD may contribute to the antidepressant effect of XCHD. In conclusion, the hyphenated method of bioactivity‐guided fractionation and LC‐ESI‐QTOFMS/MS was meaningful for the isolation and preliminary identification of the biological active components in complex matrices of traditional Chinese medicine. Copyright © 2014 John Wiley & Sons, Ltd.     

Analysis of unknown compounds in azithromycin bulk samples with liquid chromatography coupled to ion trap mass spectrometry

A selective reversed-phase liquid chromatography/mass spectrometry (LC/MS(n)) method is described for the identification of azithromycin impurities and related substances in commercial azithromycin samples. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an atmospheric pressure chemical ionization interface operated in positive ion mode. The LCQ provides on-line LC/MS(n) capability, making it ideally suited for identification purposes. In comparison with UV detection, this hyphenated technique provides as its main advantage efficient identification of novel substances without time-consuming isolation and purification procedures. Using this technique, six novel related substances detected in commercial azithromycin samples have been studied.     

On-line identification of the antifungal constituents of Erythrina vogelii by liquid chromatography with tandem mass spectrometry,ultraviolet absorbance detection and nuclear magnetic resonance spectrometry combined with liquid chromatographic micro-fractionation

In our continuing search for new antifungal agents of plant origin, the investigation of Erythrina vogelii Hook. f. (Leguminosae), a plant used in the traditional medicine of Ivory Coast to treat various infectious ailments, was undertaken. In order to rapidly identify the active principles, the crude extract was analysed by low-flow LC-1H nuclear magnetic resonance spectrometry (NMR) which gave a sensitive detection of all the main peaks. LC microfractionation was performed just after LC-NMR detection and all peaks collected were submitted to antifungal bioautography assays against Cladosporium cucumerinum. By this means, the antifungal activity could be efficiently linked to three of the LC peaks. In order to obtain complementary on-line structural information for all peaks of interest, high-resolution LC-MS-MS together with LC-UV with post-column addition of UV shifts reagents was undertaken on the crude extract. This chemical screening strategy with integrated antifungal bioassays has permitted the on-line identification of numerous constituents and has given useful information for an efficient peak-guided isolation procedure.     

Structural characterization of constituents with molecular diversity in fractions from Lysidice brevicalyx by liquid chromatography/diode-array detection/electrospray ionization tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance

A combination of electrospray ionization tandem mass spectrometry with high-performance liquid chromatography (HPLC/ESI-MSn), and hyphenation of liquid chromatography to nuclear magnetic resonance spectroscopy (HPLC/NMR), have been extensively utilized for on-line analysis of natural products, analyzing metabolite and drug impurity. In our last paper, we reported an on-line analytical method for structural identification of trace alkaloids in the same class. However, the structural types of the constituents in plants were various, such as flavanoids, terpenoids and steroids. It is important to establish an effective analytical method for on-line structural identification of constituents with molecular diversity in extracts of plants. So, in the present study, the fragmentation patterns of some isolated stilbenes, phloroglucinols and flavanoids from Lysidice rhodostegia were investigated by ESI-MSn. Their fragmentation rules and UV characteristics are summarized, and the relationship between the spectral characteristics, rules and the structures is described. According to the fragmentation rules, NMR and UV spectral characteristics, 24 constituents of different types in the fractions from L. brevicalyx of the same genus were structurally characterized on the basis of HPLC/HRMS, HPLC-UV/ESI-MSn, HPLC/1H NMR and HPLC/1H-1H COSY rapidly. Of these, six (10, 13, 14, 16, 17 and 23) are new compounds and all of them are reported from L. brevicalyx for the first time. The aim is to develop an effective analytical method for on-line structural identification of natural products with molecular diversity in plants, and to guide the rapid and direct isolation of novel compounds by chemical screening.     

Identification of highly polar nitroaromatic compounds in leachate and ground water samples from a TNT-contaminated waste site by LC-MS,LC-NMR,and off-line NMR and MS investigations

Leachate and ground water samples from a trinitrotoluene-contaminated waste disposal site near a former ammunitions plant in Stadtallendorf, Germany, were analyzed by liquid chromatography (LC)-mass spectrometry (MS) and LC-NMR hyphenated techniques to comprehensively characterize the range of highly polar nitroaromatic compounds. Wherever unknown components could not be identified by comparison with a multistandard, the spectroscopic data obtained on-line were used to make initial structure proposals, which were later confirmed by comparison with authentic reference materials. In those cases where reference materials were not commercially available, unknown compounds were isolated by HPLC cuts and their structures were elucidated by off-line NMR and MS investigations. A variety of previously unknown compounds, including nitrophenols, nitrobenzyl alcohols, methylnitrobenzoic acids, and hydroxynitrobenzoic acids, could be identified. The NMR and MS data are presented here. The main polar compounds were additionally quantified.     

On-line identification of phytochemical constituents in botanical extracts by combined high-performance liquid chromatographic-diode array detection-mass spectrometric techniques

It is necessary to determine all of the phytochemical constituents of botanical extracts in order to ensure the reliability and repeatability of pharmacological and clinical research, to understand their bioactivities and possible side effects of active compounds and to enhance product quality control. HPLC chromatographic fingerprints can be applied for this kind of documentation. Combined HPLC-diode array detection–MS techniques can provide on-line UV and MS information for each peak in a chromatogram. In most cases, direct identification of the peaks is possible, based on comparison with published data or with standard compounds. This review will primarily focus on electrospray and thermospray ionization MS and their applications for the qualitative analyses of phenolic compounds, saponins, alkaloids and other classes of natural products in botanical extracts. Twenty-one of the most commonly used herbal examples, their phytochemical analyses and characteristics of their mass spectra are described.     

Identification of Two New Phosphorylated Polyketides from a Brazilian Streptomyces sp. Through the Use of LC–SPE/NMR

Two new polyketide phosphate monoesters, phosdiecin A ( 1 ) and phosdiecin B ( 2 ), were isolated from a culture of the marine Streptomyces sp. SS99BA‐2 using the hyphenated technology LC–SPE/NMR. The compounds showed to be new representatives of an important class of antitumor antibiotic metabolites known as fostriecins. Their structures, including relative configuration attribution, were fully elucidated through extensive analyses of NMR and MALDI‐TOF/TOF HR‐MS data. Herein, the application of this system to isolate and identify the new compounds is described.     

Characterization of gingerol-related compounds in ginger rhizome (Zingiber officinale Rosc.) by high-performance liquid chromatography/electrospray ionization mass spectrometry

This study sought to determine the utility of liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) coupled with diode array detection in identifying gingerol-related compounds from crude extracts of ginger rhizome. The fragmentation behaviors of compounds in both (-)- and (+)ESI-MS/MS were used to infer and confirm the chemical structures of several groups of compounds, including the gingerols, methylgingerols, gingerol acetates, shogaols, paradols, gingerdiols, mono- and diacetyl gingerdiols, and dehydrogingerdiones. Diode array detection at different wavelengths was used to confirm MS/MS-based identification. In total, 31 gingerol-related compounds were identified from the methanolic crude extracts of fresh ginger rhizome in this study. Three of these compounds were found to be new compounds. This study demonstrated that LC/ESI-MS/MS is a powerful on-line tool for identification of gingerol-related compounds, especially for thermally labile compounds that cannot be readily detected by GC/MS analysis.     

1H and 13C NMR assignments for two lignans from the heartwood of Streblus asper

In our ongoing investigation of the bioactive constituents from plants, two new lignans, magnolignan A-2-O-beta-D-glucopyranoside and strebluslignanol were isolated from heartwood of Streblus asper, along with three known lignans, magnolignan A, magnolol, and magnaldehyde D. 1D and 2D NMR experiments, including COSY, HMQC, and HMBC, and other spectroscopic methods, including UV, IR, and MS were used for the determination of the structures and NMR assignments. Primary bioassays showed that magnolignan A-2-O-beta-D-glucopyranoside and strebluslignanol have medium cytotoxic activity against HEp-2 and HepG2 cells, with IC(50) of 13.3 microM, 46.4 microM and 10.1 microM, 21.7 microM, respectively.     

Prediction of UV and ESI-MS signal intensities

All major pharmaceutical companies maintain large collections of compounds that are used either for screening against biological targets or as synthetic precursors. The quality assessment of these compounds is typically done by liquid chromatography combined with mass spectroscopy (LC/MS) and UV purity control. To facilitate the analysis of the analytical data, we have built computational models to predict UV and MS signal intensities under experimental LC/MS conditions. The discriminant partial-least-squares technique was used for classifying compounds into those most likely to yield a MS signal and others where the signal is below the detection limit (94% and 88% correct predictions, respectively). In the case of UV prediction, we compared this statistical linear-regression technique to a knowledge-based approach. A combination of both techniques proved to be the most reliable (96/98% correct predictions of UV-active/ UV-inactive compounds). Both models have been incorporated into the automated compound integrity profiling at F. Hoffmann-La Roche.     

Screening non-colored phenolics in red wines using liquid chromatography/ultraviolet and mass spectrometry/mass spectrometry libraries

Liquid chromatography/ultraviolet (LC/UV) and mass spectrometry/mass spectrometry (MS/MS) libraries containing 39 phenolic compounds were established by coupling a LC and an ion trap MS with an electrospray ionization (ESI) source, operated in negative ion mode. As a result, the deprotonated [M-H]- molecule was observed for all the analyzed compounds. Using MS/MS hydroxybenzoic acid and hydroxycinnamic acids showed a loss of CO2 and production of a [M-H-44]- fragment and as expected, the UV spectra of these two compounds were affected by their chemical structures. For flavonol and flavonol glycosides, the spectra of their glycosides and aglycones produced deprotonated [M-H]- and [A-H]- species, respectively, and their UV spectra each presented two major absorption peaks. The UV spectra and MS/MS data of flavan-3-ols and stilbenes were also investigated. Using the optimized LC/MS/MS analytical conditions, the phenolic extracts from six representative wine samples were analyzed and 31 phenolic compounds were detected, 26 of which were identified by searching the LC/UV and MS/MS libraries. Finally, the presence of phenolic compounds was confirmed in different wine samples using the LC/UV and LC/MS/MS libraries.     

Advances in mass spectrometry-based post-column bioaffinity profiling of mixtures

In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets—pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches.     

Flavonoid Composition in the Leaves of Twelve Litsea and Neolitsea Plants

This study was aimed to study the chemodiversity of flavonoids in the Formosan Litsea and Neolitsea plants. Applications of LC‐SPE‐NMR and LC/MS hyphenated techniques in analyzing polar constituents from the leaves of L. acuminata, L. hypophaea, N. acuminatissima, and N. konishii led to the identification of 13 known flavonoids and one new flavonol dioside, quercetin 3‐O‐(2‐O‐β‐D ‐apiofuranosyl)‐α‐L ‐rhamnopyranoside. The quantity and variety of flavonoid composition in the leaves of 12 Litsea and Neolitsea plants were examined to enable more effective utilization of such bioactive ingredients. Of these, N. acuminatissima was found to contain the most quantity of flavonoids (ca. 0.24%leaves).     

Rapid identification and evaluation of antioxidant compounds from extracts of Petasites japonicus by hyphenated-HPLC techniques

Gradient HPLC coupled to Diode Array Detector (DAD), MS/MS and NMR was applied to the rapid structure determination of major compounds of methanol extracts from leaves and roots of Petasites japonicus. The relative antioxidant capacities of the compounds were evaluated by an HPLC system with post-column on-line antioxidant detection based on 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical scavenging. Six compounds were successfully separated on a reverse-phase C(18) column and were identified as 5-caffeoylquinic acid (5-CQA), fukinolic acid (FA), 3,5-di-O-caffeoylquinic acid (3,5-DCQA), quercetin-3-O-(6″-acetyl)-β-glucopyranoside (QAG), 4,5-di-O-caffeoylquinic acid (4,5-DCQA) and kaempferol-3-O-(6″-acetyl)-β-glucopyranoside (KAG) by MS/MS and (1)H NMR data. Among these compounds, those containing a caffeoyl moiety (5-CQA, FA, 3,5- and 4,5-DCQA) showed relatively strong radical scavenging capacity, with 3,5-DCQA having the greatest radical scavenging capacity in leaf (23.09% of total antioxidant capacity) and root (26.47%) extracts. The relative radical scavenging portion of QAG was only 3.41% in the leaves and KAG did not show any radical scavenging activity. These results demonstrate that the hyphenated HPLC techniques can be successfully applied to rapidly identify structures and evaluate antioxidant activities without prior purification of compounds from plant tissues of P. japonicus.     

Rapid analysis of antibiotic-containing mixtures from fermentation broths by using liquid chromatography-electrospray ionization-mass spectrometry and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry

A crucial step in the isolation of antibiotic substances is establishing whether or not the isolated material represents a new chemical entity. Because of the importance of molecular weight to this process--known as dereplication--mass spectrometry has traditionally played an active role. In this communication a strategy for utilizing liquid chromatography-mass spectrometry (LC/MS) for novelty assessment is described. Crude extracts (20-50 μg) are chromatographed by conventional bore high-performance liquid chromatography (1 mL/min) after which a postcolumn split to divert roughly one-tenth of the sample to the mass spectrometer for molecular weight determination by electrospray ionization (ESI) mass spectrometry. The majority of the effluent is sent to a UV detector and ultimately collected as 1-min fractions for biological testing. As a secondary confirmation of molecular weight, an aliquot of each fraction (< 5%) is taken for analysis by matrix-assisted laser desorption ionization (MALDI). The improved efficiency of this approach over more traditional schemes utilizing off-line fraction collection and conventional ionization methods can be explained by several factors. First, the superior sensitivity of ESI and MALDI means that less material is required for successful analysis. Second, on-line LC/MS optimizes the efficiency of sample transfer and saves both time and labor. Furthermore, the concentration dependence of ESI allows a majority of the material injected for LC/MS to be recovered for biological testing without compromising the signal available for molecular weight determination. As a validation of the above method, crude extracts containing two well-characterized antibiotics--teicoplanin and phenelfamycin--were examined. Results from these analyses are presented along with data from the analysis of a potent unknown antifungal sample.     

Sequencing of bacitracin A and related minor components by liquid chromatography/electrospray ionization ion trap tandem mass spectrometry

A selective reversed-phase liquid chromatography/mass spectrometry (LC/MS(n)) method is described for the characterization of related compounds in commercial bacitracin samples. Mass spectral data for these polypeptide antibiotics were acquired on a LCQ ion trap mass spectrometer equipped with an electrospray ionization probe operated in the positive and negative ion mode. The LCQ ion trap is ideally suited for the sequencing of those linear side-chain cyclized peptides because it provides on-line LC/MS(n) capability. Using this method bacitracin A, 1-epibacitracin A, bacitracins B(1), B(2), B(3) and bacitracin F were sequenced and previous sequencing was confirmed. Bacitracins C(1), C(2), C(3), D, H(2) and H(3) were resolved chromatographically and their ring portion was sequenced for the first time. Four components not described in the literature (1-epibacitracin B(1), 1-epibacitracin B(2), 1-epibacitracin C(1) and H(4)) were sequenced completely for the first time. The main advantage of this hyphenated LC/MS(n) technique is the characterization of the related substances without time-consuming isolation and purification procedures.