Flavonoid O-glycosides Research Articles

Comprehensive Profiling and Inheritance Patterns of Metabolites in Foxtail Millet.

Metabolomics aims at determining a sample's metabolites profile and hence provides a straight functional statement of an organism's physiological condition. Here, we investigated comprehensive profiling, natural variation and species-specific accumulation of both primary and secondary metabolites in foxtail millet using LC-MS, and inheritance patterns of metabolome in millet hybrids. The application of a broad target metabolomics method facilitated the simultaneous identification and quantification of more than 300 metabolites. The metabolic analysis of these compounds, such as flavonoids, phenolamides, hydrocinnamoyl derivatives, vitamins and LPCs, revealed their developmentally controlled accumulation, and natural variation in different tissues/varieties. Species-specific accumulation of secondary metabolites was observed based on a comparative metabolic analysis between millet and rice, such as flavonoid O-rutinosides/neohesperidosides and malonylated flavonoid O-glycosides. In analyzing the metabolic variations between hybrid progenies and their parental lines, including a photothermo-sensitive genic male sterility line and five Zhangzagu varieties, metabolic overdominant, and dominant patterns of inheritance could be observed. For example, hydrocinnamoyl derivatives and feruloylated flavonoids were identified as over-parent heterosis (overdominant) metabolites in milet hybrids. Our work paves the way for developing predictors of hybrid performance and the future analysis of the biosynthesis and regulation of relevant metabolic pathways in millet.

LC-ESI/LTQOrbitrap/MS/MSn Analysis Reveals Diarylheptanoids and Flavonol O-glycosides in Fresh and Roasted Hazelnut (Corylus avellana cultivar “Tonda di Giffoni”)

Hazelnut represents one of the most consumed food, containing a large range of bioactive and health-promoting compounds. Investigation of the fatty component of C. avellana nut has been widely carried out, but little is known about its phenolic content. In order to achieve deeper insight into the polar fraction of the n-butanol extracts of both fresh and roasted kernels of C. avellana cultivar, “Tonda di Giffoni” a PGI (Protected Geographical Indication) product of Campania region (Italy), a phytochemical investigation was carried out. High performance liquid chromatography coupled to electrospray ionization, multiple-stage linear ion-trap and orbitrap high-resolution mass spectrometry (LC-ESI/LTQOrbitrap/MS/MSn) profile of the fresh and roasted hazelnut led to the identification of eleven phenolic compounds corresponding to diarylheptanoid derivatives, flavonoid O-glycosides and ellagic acid. Moreover, the antioxidant activity of isolated compounds was evaluated by TEAC assay which showed that ellagic acid and flavonoid derivatives possessed the highest free-radical-scavenging activity.

Enrichment and Purification of Total Ginkgo Flavonoid O-Glycosides from Ginkgo Biloba Extract with Macroporous Resin and Evaluation of Anti-Inflammation Activities In Vitro.

In the present study, the performance and separation characteristics of six macroporous resins for the enrichment and purification of total ginkgo flavonoid O-glycosides (TGFs) (quercetin (I), kaempferol (II), isorhamnetin (III)) from Ginkgo Biloba extracts (EGB) are evaluated. The adsorption and desorption properties of TGFs are studied on macroporous resins, including D101, D201, AB-8, HPD400, D301, and D311. Along with the results, AB-8 resin exhibits the best adsorption and desorption capacity for these three ginkgo flavonoid O-glycosides among the six resins. Adsorption isotherms are created on AB-8 resin and fit well to the Langmuir (R2 > 0.96) and Freundlich (R2 > 0.92, 0.3 < 1/n < 0.7) models. After the treatment with gradient elution on AB-8 resin packed chromatography column, the contents of the three main ginkgo flavonoid O-glycosides (I, II, and III) increase from 8.93%, 9.88%, and 6.11% in the extracts to 30.12%, 35.21%, and 14.14%, respectively, in the product. The recoveries of compounds I, II, and III are 88.76%, 93.78%, and 60.90%, respectively. Additionally, the anti-inflammatory effects of TGFs are evaluated in LPS-treated RAW 264.7 macrophages, and the result demonstrates that TGFs could significantly inhibit LPS-induced NO release in vitro in a dose-dependent manner compared with the control group. These findings suggest that TGFs could potentially be natural antioxidants and anti-inflammatory ingredients that could be used in pharmaceutical products and functional food additives.

Simultaneous identification and determination of flavonoids in Dendrobium officinale

BackgroundThe quality of material medicine resources has had a considerable impact on the development of the health industry, which has created a bottleneck for traditional Chinese medicine (TCM). Dendrobium officinale, which has been widely used for health prevention in TCM, has become a high-nutritive health food that is strongly recommended by many white-collar workers and people who pay more attention to their health. The aim of this study was to develop a method to authenticate and evaluate D. officinale from different origins via simultaneous qualitative and quantitative analyses of flavonoid glycosides. Ultra-high-performance liquid chromatography-electrospray ionization/mass spectrometry was used for the structural elucidation of the compounds.Results9 characteristic peaks, including those representing 7 flavonoid C-glycosides and 2 flavonoid O-glycosides, were identified. Additionally, the contents of 5 representative flavonoid glucosides in 25 batches of D. officinale from different sources were determined. To further investigate the different sources of the 25 batch samples, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were carried out. A study on the methodology revealed that all results were reliable.ConclusionsThis method is an efficient tool for the rapid identification of the different geographical origins of D. officinale and provides references for the quality evaluation of other natural products.

Permeability through the Caco-2 cell monolayer of 42 bioactive compounds in the TCM formula Gegen-Qinlian Decoction by liquid chromatography tandem mass spectrometry analysis

Caco-2 cell monolayer model was used to evaluate the intestinal permeability of 42 bioactive compounds in the famous traditional Chinese medicine (TCM) formula Gegen-Qinlian Decoction (GQD). These compounds include alkaloids, flavonoids and glycosides, triterpenoid saponins, and coumarins. Their transportations across the cell monolayers in the forms of herb extract and formula extract were monitored by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) analysis. Most alkaloids from Huang-Lian; flavonoid C-glycosides from Ge-Gen and Huang-Qin; O-glycosides from Ge-Gen, Huang-Qin and Gan-Cao; O-glucuronides from Huang-Qin; and coumarins from Gan-Cao exhibited favorable permeability. Their PAB values were >1.05×10−5cm/s, and efflux ratios (ER, PBA/PAB) were≤1.0. In contrast, triterpenoid saponins showed poor permeability (PAB≤1.50×10−6cm/s, ER≤1.5), indicating a paracellular diffusion mechanism. Furthermore, GQD could remarkably improve the intestinal transport of alkaloids in Huang-Lian, flavonoid C-glycosides in Ge-Gen, as well as coumarins and flavonoid O-glycosides in Gan-Cao. These results indicate herb–herb interactions in GQD.

Flavonoids of Lotus (Nelumbo nucifera) Seed Embryos and Their Antioxidant Potential.

Flavonoids from lotus (Nelumbo nucifera) seed embryos were fractionated over a macroporous resin chromatography into 2 main fractions (I and II), and subsequently identified by high-performance liquid chromatography coupled with tandemmass spectrometry (HPLC-MS2 ). Sixteen flavonoids were identified in lotus seed embryos, including 8 flavonoid C-glycosides and 8 flavonoid O-glycosides, in which the flavonoid C-glycosides were the main flavonoids. Among them, 2 flavonoid O-glycosides (luteolin 7-O-neohesperidoside and kaempferol 7-O-glucoside) were identified in lotus seed embryos for the 1st time. For further elucidating the effects of flavonoid C-glycosides to the bioactivities of lotus seed embryos, we compared the differences of the flavonoids and their antioxidant activities between leaves and seed embryos of lotus using the same methods. The results showed the antioxidant activity of flavonoids in lotus seed embryos was comparable or higher than that in lotus leaves, whereas the total flavonoid content in seed embryos was lower than lotus leaves which only contained flavonoid O-glycosides. The flavonoid C-glycosides of lotus seed embryos had higher antioxidant properties than the flavonoid O-glycosides presented in lotus leaves. This study suggested that the lotus seed embryos could be promising sources with antioxidant activity and used as dietary supplements for health promotion.

An enhanced targeted identification strategy for the selective identification of flavonoid O-glycosides from Carthamus tinctorius by integrating offline two-dimensional liquid chromatography/linear ion-trap-Orbitrap mass spectrometry, high-resolution diagnostic product ions/neutral loss filtering and liquid chromatography-solid phase extraction-nuclear magnetic resonance

Targeted identification of potentially bioactive molecules from herbal medicines is often stymied by the insufficient chromatographic separation, ubiquitous matrix interference, and pervasive isomerism. An enhanced targeted identification strategy is presented and validated by the selective identification of flavonoid O-glycosides (FOGs) from Carthamus tinctorius. It consists of four steps: (i) enhanced separation and detection by offline two-dimensional liquid chromatography/LTQ-Orbitrap MS (offline 2D-LC/LTQ-Orbitrap MS) using collision-induced dissociation (CID) and high-energy C-trap dissociation (HCD); (ii) improved identification of the major aglycones by acid hydrolysis and LC-SPE-NMR; (iii) simplified spectral elucidation by high-resolution diagnostic product ions/neutral loss filtering; and (iv) more convincing structural identification by matching an in-house library. An offline 2D-LC system configuring an Acchrom XAmide column and a BEH Shield RP-18 UPLC® column enabled much better separation of the easily co-eluting components. Combined use of CID and HCD could produce complementary fragmentation information. The intensity ratios of the aglycone ion species ([Y0-H]−/Y0− and [Y0-2H]−/Y0−) in the HCD-MS2 spectra were found diagnostic for discriminating the aglycone subtypes and characterizing the glycosylation patterns. Five aglycone structures (kaempferol, 6-hydroxykaempferol, 6-methoxykaempferol, carthamidin, and isocarthamidin) were identified based on the 1H-NMR data recorded by LC-SPE-NMR. Of the 107 characterized flavonoids, 80 FOGs were first reported from C. tinctorius. Unknown aglycones, pentose, and novel acyl substituents were discovered. A new compound thereof was isolated and fully identified, which could partially validate the MS-oriented identification. This integral strategy can improve the potency, efficiency, and accuracy in the detection of new compounds from medicinal herbs and other natural sources.

Rapid separation and identification of multiple constituents in Danhong Injection by ultra-high performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry

To characterize and identify multiple constituents in Danhong injection (DHI), a fast ultra-high performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-QTOF/MS) method was established and validated in the present study. A total of 63 compounds, including 33 phenolic acids, 2 C-glycosyl quinochalcones, 6 flavonoid O-glycosides, 4 iridoid glycosides, 6 organic acids, 5 amino acids, and 3 nucleosides, were identified or tentatively characterized. In conclusion, the UHPLC-ESI-QTOF/MS method is useful and efficient for in-depth structural elucidation of chemical compounds in complex matrices of herbal medicines such as DHI.

MALDI-MS of flavonoids: a systematic investigation of ionization and in-source dissociation mechanisms.

Matrix assisted laser desorption ionization (MALDI) is a technique widely employed in the analysis of proteins and peptides, and nowadays it has also been applied to small molecules. There is little significant information regarding the in-source dissociation processes on MALDI for natural products. Twenty-six flavonoids (flavanones, flavones and flavonols) were analyzed by MALDI using different methods (with different matrices) and without matrix to comprehend the in-source reactions and establish good analysis methods for these compounds. Depending on the class, structure and the laser intensity applied, methoxylated flavonoid aglycones can eliminate methyl radicals (˙CH3) in the source, such as flavonols, but lithium 2,4-dihydroxybenzoate matrix suppresses the ˙CH3 eliminations and retro-Diels-Alder cleavages in the source. All of the flavonoid O-glycosides evaluated herein eliminated the sugar in source, even in the presence of the matrix, and its product radical ions ([M-H-sugar](-)˙) were observed in the negative mode. The flavone C-glycosides suffered intense dissociation, which was reduced by the addition of a matrix and the application of low laser intensity, mainly in the negative mode. Depending on the hydroxyl substituents, the [M-H-H](-)˙ ion was observed with variable relative intensity in the spectra.

Comprehensive profiling and natural variation of flavonoids in rice

Flavonoids constitute a major group of plant phenolic compounds. While extensively studied in Arabidopsis, profiling and naturally occurring variation of these compounds in rice (Oryza sativa), the monocot model plant, are less reported. Using a collection of rice germplasm, comprehensive profiling and natural variation of flavonoids were presented in this report. Application of a widely targeted metabolomics method facilitated the simultaneous identification and quantification of more than 90 flavonoids using liquid chromatography tandem mass spectrometry (LC-MS/MS). Comparing flavonoid contents in various tissues during different developmental stages revealed tissue-specific accumulation of most flavonoids. Further investigation indicated that flavone mono-C-glycosides, malonylated flavonoid O-hexosides, and some flavonoid O-glycosides accumulated at significantly higher levels in indica than in japonica, while the opposite was observed for aromatic acylated flavone C-hexosyl-O-hexosides. In contrast to the highly differential accumulation between the two subspecies, relatively small variations within subspecies were detected for most flavonoids. Besides, an association analysis between flavonoid accumulation and its biosynthetic gene sequence polymorphisms disclosed that natural variation of flavonoids was probably caused by sequence polymorphisms in the coding region of flavonoid biosynthetic genes. Our work paves the way for future dissection of biosynthesis and regulation of flavonoid pathway in rice.

ChemInform Abstract: Glycosylation Reactions in the Synthesis of Flavonoid Glycosides

AbstractReview: [82 refs.

Glycosylation Reactions in the Synthesis of Flavonoid Glycosides

An overview of flavonoid glycoside synthesis has been provided with an emphasis on the construction of the glycosidic linkages. 1 Introduction 2 Synthetic Tactics 3 Synthesis of Flavonoid O-Glycosides 3.1 Glycosylation with Glycosyl Bromides 3.2 Glycosylation with Glycosyl Imidates 3.3 Glycosylation with Glycosyl Esters 3.4 Glycosylation with Glycosyl Fluorides 3.5 Combined Application of Different Types of Glycosyl Donors­ 4 Synthesis of Flavonoid C-Glycosides 5 Synthesis of Flavonoid Mixed O,C-Glycosides 6 Conclusion

Bioactive phenolics of fresh and freeze-dried sweet and semi-spicy pepper fruits (Capsicum annuum L.)

Changes in contents of eight phenolic compounds in extracts from fresh and freeze-dried placentas and pericarps of four cultivars of pepper fruit (Capsicum annuum) were investigated. These compounds comprised two phenolic acid derivatives, four flavonoid O-glycosides, and two flavonoid C-glycosides. Quantitative HPLC–DAD analysis was performed on the basis of the peak area of standards isolated from pepper and identified by MS/MS2 analysis. The main compounds in the pericarp were quercetin O-glycosides, while trans-p-feruloyl-β-d-glucopyranoside predominated in the placenta, at a concentration almost 10 times higher than that in the pericarp. In general, lower levels of phenolic acid derivatives were found in extracts from lyophilised fruits, but different directions of changes in the concentrations of flavonoid O- and C-glycosides from fresh and freeze-dried fruits were noted. The antioxidant activity measured by the DPPH method was higher for samples of fresh fruits than for freeze-dried samples. Additionally, the antioxidant activities of extracts from placentas correlated well with the changes in flavonoid C-glycosides (R2=0.63), while the antioxidant activities from the pericarp correlated with changes in phenolic acid derivatives (R2=0.78).

Low energy induced homolytic fragmentation of flavonol 3‐O‐glycosides by negative electrospray ionization tandem mass spectrometry

Negative ESI-QIT-MS of several subtypes of flavonoid O-glycosides is known to produce deprotonated molecular ions which undergo homolytic fragmentation at the glycosidic bond upon collision-induced dissociation (CID). However, these subtypes have never been simultaneously compared under unified MS conditions. The (-)-ESI-MS(n) fragmentations of 69 flavonoid O-glycosides, involving eight subtypes, were analyzed using a quadrupole ion-trap mass spectrometer with collision energies varying from 18-44%. Factors influencing the homolytic glycosidic bond fragmentation, such as collision energy, hydroxylation of aglycone, and glycosylation pattern, were comprehensively studied. Under the unified CID-QIT-MS(2) conditions, the precursor deprotonated molecular ions [M-H](-) for 3-O-glycosyl, 3,7-di-O-glycosyl and 3,6,7-tri-O-glycosyl flavonols experienced homolytic fragmentation at the glycosidic bond and generated the radical aglycone ion [Y0-H](-•). This gas-phase CID fragmentation behavior was not observed for the other subtypes. A general trend was found that hydroxyl substitution at C-6, glycosylation at C-6/C-7, and acetylation of the saccharide moiety remarkably suppressed this fragmentation. In addition, flavonol 3-O-diglycosides (disaccharides) possessing a 1 → 2 glycosidic bond generated more abundant [Y0-H](-•) product ions than those with a 1 → 3 or 1 → 6 bond. The terminal sugar triggered the homolytic fragmentation in the order Rha > Xyl > Glc. Moreover, new counterexamples were found for previously reported fragmentation rules. The low-energy CID homolytic fragmentation was diagnostic for structural identification of flavonol 3-O-glycosides. We have summarized key factors affecting this fragmentation. The results could be useful for rapid characterization of flavonoid O-glycosides in complicated herbal extracts.

Fuzhuanins A and B: The B-ring Fission Lactones of Flavan-3-ols from Fuzhuan Brick-Tea

Fuzhuan brick-tea is a special dark tea prepared from the leaves of Camellia sinensis var. sinensis. Its production involves a fungal fermentation stage, which forms the unique flavors and functions by a series of biochemical reactions. Our phytochemical research of the material led to the isolation of two new B-ring fission lactones of flavan-3-ols, fuzhuanins A (1) and B (2). In addition, three other flavan-3-ol derivatives (3-5), three flavone C-glycosides (6-8), eight flavonoid O-glycosides (10-17), five simple phenolics (19-23), two norisoprenoid glycosides (24, 25), two sesquiterpenoids (26, 27), and theobromine (28), as well as two flavonoid anions (9 and 18), were also identified. The structures of these compounds were determined by spectroscopic methods. Compounds 4, 19, 20, 22-24, 26, and 27 were reported for the first time in Camellia spp. and tea. Furthermore, HPLC analysis method was performed to compare the chemical constituents of the before/after fungal fermentation Fuzhuan brick-teas. Compound 1 was indicated as one of the major characteristic constituents generated in the fungal fermentation process. The IC50 value of the antiproliferative activity of 2 on HeLa cells was assayed as 4.48 μM. None of the isolated compounds showed any inhibition activity against the enteric pathogenic microbes at 800 μg/mL by the hole plate diffusion method.

UPLC–PDA–ESI–MS/MS Analysis of Compounds Extracted by Cardiac h9c2 Cell from Polygonum orientale

A flavonoid-enriched extract (FEE) of Polygonum orientale was reported to show cardioprotective effect but only very few compounds were reported to contribute to the effect. Identification of compounds interacting with the target cardiac cell is important for the understanding of active compounds. To develop an efficient method for the screening of potential active compounds directly acting on the target cardiac cell in FEE and to structurally characterise these compounds. Flavonoid-enriched extract was prepared by extraction of the plant with water, addition of ethanol to the solution to remove polysaccharides and proteins, and removal of tannins by a polyamide column chromatography. Cell extraction was conducted on a cardiac h9c2 cell and the solution containing compounds released from the cell were desalted by solid phase extraction. Compounds present in the cell extract were detected by ultra-performance liquid chromatography (UPLC) and targeted multi-reaction monitoring (MRM), while their structures were characterised by UPLC-photodiodide array (PDA)-electrospray ion source (ESI)-MS/MS investigations of the FEE. Twenty-three potentially active phenolics including ten flavonoid C-glycosides and six flavonoid O-glycosides have been identified from the 40 compounds screened in the cell extract. Among these compounds, three were new and nine were identified from this plant for the first time. Strategies for the structural characterisation of flavonoid glycosides were also discussed. The study has shown that FEE contains the flavonoid as its major principles and the coupling of UPLC-PDA-ESI-MS/MS and targeted UPLC-MRM with target cell extraction is an efficient method for the screening and structural characterisation of potential active compounds.

Identification of common glycosyl groups of flavonoid O‐glycosides by serial mass spectrometry of sodiated species

Flavonoid O-glycosides are a ubiquitous and important group of plant natural products in which a wide variety of sugars are O-linked to an aglycone. Determining the identity of the sugars, and the manner in which they are linked, by mass spectrometry alone is challenging. To improve the identification of common O-linked di- and trisaccharides when analysing mixtures of flavonoid O-glycosides by liquid chromatography/mass spectrometry (LC/MS), the fragmentation of electrosprayed sodium adducts in an ion trap mass spectrometer was investigated. The sodium adducts [M + Na](+) of kaempferol 3-O-glycosides generated sodiated glycosyl groups by the neutral loss of kaempferol. The product ion spectra of these sodiated glycosyl groups differed between four isomeric kaempferol 3-O-rhamnosylhexosides and four isomeric kaempferol 3-O-glucosylhexosides in which the primary hexose was either glucose or galactose and bore the terminal glucose or rhamnose at either C-2 or C-6. Fragmentation of sodiated glycosyl groups from linear O-triglucosides and branched O-glucosyl-(1 → 2)-[rhamnosyl-(1 → 6)]-hexosides produced sodiated disaccharide residues, and the product ion spectra of these ions assisted the identification of the complete sugar. The product ion spectra of the sodiated glycosyl groups were consistent among flavonoid O-glycosides differing in the position at which the sugar was O-linked to the aglycone, and the nature of the aglycone. The abundance of sodiated species was enhanced by application of a pre-trap collision voltage, without the need to dope with salt, allowing automated LC/MS methods to be used to identify the glycosyl groups of common flavonoid O-glycosides, such as rutinosides, robinobiosides, neohesperidosides, gentiobiosides and sophorosides.

Identification of chemical constituents in extracts and rat plasma from Fructus Aurantii by UPLC‐PDA‐Q‐TOF/MS

Fructus Aurantii, as a component of several compound formulae, shows many bioactivities. As is well known, the constituents of traditional Chinese medicines are very complex and multiple constituents are responsible for the therapeutic effect. However, the concrete compounds are difficult to confirm. Therefore, studies on the constituents absorbed into serum and their metabolites are necessary. To search for the active constituents in Fructus Aurantii. An ultra-performance liquid chromatography-photodiode array detector-quadrupole time-of-flight mass spectrometry (UPLC-PDA-Q-TOF/MS) method was established to analyse the aqueous extract in Fructus Aurantii and the constituents absorbed into blood. Compound identification was made by matching the empirical molecular formula with those of the reported compounds and UV spectra, and further elucidated by lower energy CID mass spectra. Twelve flavonoid O-glycosides were detected, and nine compounds were tentatively identified as polymethoxylated flavones. Six parent compounds were identified and four metabolites were observed in rat plasma, two of which were identified as naringenin glucuronide and hesperetin glucuronide, respectively. The approach developed has proved useful in the study of the active constituents in traditional Chinese medicines.

Phytochemical Composition and Antioxidant Activity ofLaurus nobilisL. Leaf Infusion

Laurus nobilis L. (laurel) leaves are frequently used as a spice for cooking purposes. Folk medicine in many countries uses the infusion of the plant in stomachic and carminative remedies, as well as for the treatment of gastric diseases. Little information is available about the phytochemical composition of the infusion of dried leaves, which is a way to consume this aromatic and medicinal plant. Phytochemical investigations on the infusion were performed by high-performance liquid chromatography (HPLC) with a diode array detector (DAD) and direct electrospray ionization-tandem mass spectrometry. Several flavonoid derivatives were detected. Semipreparative HPLC from the infusion of laurel leaves isolated 10 flavonoid O-glycosides, one flavonoid C-glycoside, catechin, and cinnamtannin B1. Structures of the isolated compounds were computed on the basis of spectral measurements including high-resolution mass spectrometry spectroscopy and one- and two-dimensional nuclear magnetic resonance techniques. The amount of the flavonoids was also determined by HPLC-DAD. The antioxidant activity of the tea and the isolated compounds was also measured using two different in vitro methods: the Briggs-Rauscher oscillating reaction test, at a pH similar to that of the gastric juice, and the Trolox equivalent antioxidant capacity assay, at the pH of blood. For the infusion and the methanol extract the total phenolic content was also measured using the Folin-Ciocalteu reagent.

Rapid separation and identification of 54 major constituents in Buyang Huanwu decoction by ultra‐fast HPLC system coupled with DAD‐TOF/MS

Buyang Huanwu Decoction (BYHWD), is a well-known traditional Chinese preparation consisting of Radix Astragali, Radix Angelicae Sinensis, Rhizoma Ligustici Chuanxiong, Radix paeoniae Rubra, Flos Carthami, Semen Persicae and Lumbricus. An ultra-fast high-performance liquid chromatography (HPLC) method coupled with diode array detection (DAD) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS) has been developed for rapid separation and structural identification of constituents in BYHWD. Using an ultra-fast HPLC system with short columns (4.6 x 50 mm, 1.8 microm), the total analysis time for this complex prescription is less than 30 min. With various fragmentor voltages in TOF/MS, accurate mass measurements (less than 5 ppm error) for molecular ions and characteristic fragment ions could represent reliable identification criteria for these compounds. Fifty-four major constituents from BYHWD sample, including four C-glycosyl quinochalcones, four flavonoid O-glycosides, sixteen isoflavones, six monoterpene glycosides, eight saponins, four organic acids and five amino acids, were identified or tentatively characterized based on their retention times, DAD and TOF/MS data. All the compounds were further assigned in the seven individual crude drugs. In conclusion, the ultra-fast HPLC with DAD-TOF/MS is a highly useful and efficient technique to separate and identify constituents in complex matrices of herbal medicines or preparations.