Application of achiral-chiral two-dimensional HPLC for separation of phenylalanine and tryptophan enantiomers in dietary supplement.

This study developed a two-dimensional heart-cutting LC method for the separation of amino acid enantiomers. Two approaches for achiral separation of amino acids, phenylalanine and tryptophan, were selected. Amino acids were separated on C18 or hydrophilic interaction liquid chromatography (HILIC) columns in first dimension after their enantiomer separation on a teicoplanin chiral column in second dimension. Mobile phases for both separation systems were optimized by testing different types of organic modifiers, concentrations of ion-pair agent (sodium 1-octanesulfonate), and ionic modifier (ammonium acetate). The resolution of enantiomers higher than 1.5 for both amino acids was achieved using a C18-teicoplanin coupled column separation system with mobile phases methanol/2 mM sodium 1-octanesulfonate (10:90 and 75:25, step gradient between achiral and chiral columns, respectively). The lower resolution of amino acid enantiomers (RS ˃ 0.9), but higher column efficiency, was achieved on a HILIC-teicoplanin separation system with mobile phases acetonitrile/50 mM ammonium acetate (90:10 and 80:20, step gradient between achiral and chiral columns, respectively). The developed heart-cutting 2D-LC methods were validated in terms of linearity, limit of detection, limit of quantification, precision, and accuracy. The results suggested that the developed methods were applicable for the simultaneous determination of amino acid enantiomers in the dietary supplement. The sample contained l-phenylalanine and l-tryptophan.

Isolation of bioactive components with soluble epoxide hydrolase inhibitory activity from Stachys sieboldii MiQ. by ultrasonic-assisted extraction optimized using response surface methodology.

Stachys sieboldii MiQ (SSM) is an important food and medicinal herb in Korea, used to improve memory of patients with senile dementia and cardiovascular diseases. However, little information on bioactive components from SSM or standardized extraction methods for these components is available. This study isolated and purified major components from SSM for the first time, and assessed their ability to inhibit soluble epoxide hydrolase (sEH). The results showed that acteoside is the most potent inhibitor of sEH, with an IC50 of 33.5 ± 0.5 µM. Additional active components, including harpagide, tryptophan, and 8-acetate-harpagide, along with acteoside, were tentatively identified using high-performance liquid chromatography photodiode array tandem mass spectrometry (HPLC-PDA-MS/MS) and quantified using an ultraviolet detector at 210 nm. Further, an ultrasonic-assisted extraction technique for extraction of four bioactive compounds in SSM was developed and optimized using response surface methodology (RSM). The optimal extraction conditions were: extraction time, 30.46 minutes; extraction temperature, 67.95 °C, and methanol concentration 53.85%. The prediction model of RSM was validated with laboratory experiments. The similarity between predicted and actual values was 97.84%. The extraction method is thus a rapid, environment-friendly, energy-saving method can be applied to extract bioactive components from SSM in large quantities.

Determination of Tryptophan Metabolites in Serum and Cerebrospinal Fluid Samples Using Microextraction by Packed Sorbent, Silylation and GC-MS Detection.

Indole-containing acids-tryptophan metabolites-found in serum and cerebrospinal fluid (CSF) samples of patients with diseases of the central nervous system (CNS) were determined with the use of microextraction by packed sorbent (MEPS) followed by silylation and gas chromatography-mass spectrometry (GC-MS) analysis. MEPS with the following silylation led to the reproducible formation of derivatives with an unsubstituted hydrogen ion in the indole ring, the chromatographic peaks of which are symmetric and can be used for GC-MS analysis without additional derivatization. The recoveries of analytes at the limit of quantitation (LOQ) levels were 40-80% for pooled CSF and 40-60% for serum. The limit of detection (LOD) and LOQ values were 0.2-0.4 and 0.4-0.5 µM, respectively, for both CSF and serum. The precision (the reproducibility, RSD) value of less than 20% and the accuracy (the relative error, RE) value of less than ±20% at the LOQ concentrations meet the Food and Drug Administration (FDA) recommendations. Linear correlations for all analytes were determined over a potentially clinically significant range of concentrations (0.4-10 µM for serum, R2 ≥ 0.9942, and 0.4-7 µM for CSF, R2 ≥ 0.9949). Moreover, MEPS significantly reduced the matrix effect of serum compared to liquid-liquid extraction (LLE), which was revealed in the example of reducing the amount of cholesterol and its relative compounds.

Enantioseparation of tryptophan and its unnatural derivatives by nano-LC on CSP-teicoplanin silica based.

This work deals with the potentiality of nano liquid chromatography (Nano-LC) for the chiral separation of racemic mixture of tryptophan and some selected derivatives by using 100 µm i.d. fused silica capillary packed with teicoplanin bonded to 5 µm diol silica stationary phase. The experiments were carried out by using a cheap and laboratory-assembled nano-LC-UV system. Elution was done in an isocratic mode using a polar organic mobile phase. In order to find the optimum chiral separation of the studied enantiomers, some chromatographic experimental parameters were systematically studied and optimized. Among them, mobile phase composition, namely organic modifier type and concentration, buffer type and pH and aqueous content and sample solvent dilution on retention time, retention factor and enantioresolution factor were studied. Baseline enantioresolution and good peak shape was achieved utilizing the mobile phase containing 40 mM ammonium formate at pH pH 2.5 in ACN/water/acetone (60:30:10, v/v/v) at 520 nL/min in less than 8 min analysis time.

Targeted expression and purification of fluorine labelled cold shock protein B by using an auxotrophic strategy.

High resolution NMR spectroscopy is a seminal method in modern structural biology to obtain insights into proteins' structure, dynamics and function at dilute condition as well as in a cell-like environment or even intracellularly. Usually, 1H, 15N or 13C nuclei are predominantly used for the characterization of the protein of interest. These measurements are limited due to the wealth of chemical shifts and background signals arising from all molecules present in the NMR test tube. On top of that, the protein under study has to be isotopically enriched in nitrogen and/or carbon nuclei enabling to overcome the inherently low natural abundance of 13C and 15N NMR active isotopes. In this way switching to 19F NMR spectroscopy strongly reduces the total amount of signals seen in an NMR spectrum as it turns off background signals and is for this reason extremely attractive for highly-resolved investigations of proteins performance measured directly in cells or in a cell-like environment. Here we show the effective expression and purification of cold shock protein B from Bacillus subtilis (BsCspB) using fluorine labelled phenylalanine or fluorine labelled tryptophan residues. We reveal that fluorine labelled BsCspB represents the same fold on a secondary as tertiary level as seen for the wild type protein independent of the labelling position illuminating the soft character of fluorine insertion. This experimental setup of targeted fluorine labelling sets a profound ground for a broad range of highly-resolved 19F NMR applications to be performed in a complex cellular environment.

Investigation of the interaction between serotonin-related compounds and phenylboronate moieties in monolithic silica disk-packed spin columns.

Solid-phase extraction technologies are widely used for sample pretreatment in bioanalysis. Monolithic silica disk-packed spin columns modified with phenylboronate moieties have been developed for the selective extraction of cis-diol compounds such as catecholamines. However, in our preliminary studies, serotonin was found to also be extracted in this treatment, along with catecholamines. In this study, the interaction between serotonin-related compounds (serotonin, tryptophan, 5-hydroxy-tryptophan and 5-hydroxyindoleacetic acid) and phenylboronate moieties was investigated. We found that only serotonin was extracted with phenylboronate-modified monolithic silica, whereas tryptophan, 5-hydroxy-tryptophan and 5-hydroxyindoleacetic acid were not. Hydrophobic interactions rather than ionic interactions were the primary factor for the adsorption of serotonin to phenylboronate. Finally, the selective pretreatment procedure for catecholamines was improved: thus, the method could be applied for the pretreatment of bio-samples.

Asperorydines A-M: Prenylated Tryptophan-Derived Alkaloids with Neurotrophic Effects from Aspergillus oryzae.

As part of our program to discover new bioactive agents from fungi, 13 new alkaloids accompanying 13 known related alkaloids were isolated from a wild strain of Aspergillus oryzae L1020. Compounds 1 and 2 have unprecedented 6/6/5/7/5 and 6/6/6/5/5 chemical skeletons, representing new members of quinoline alkaloids. Compound 3 is a new macrolactam with an unusual 6/5/6/8 ring system. Compounds 4-13 are new α-cyclopiazonic acid-related alkaloids. The absolute configurations of 1-4, 8, and 9 were assigned by electronic circular dichroism calculations. Compounds 2, 5, 6, 11, 14, 22, and 26 exhibit pronounced neurite outgrowth-promoting effects on PC12 cells in the range of 25-100 µM.

Hydrophobic Amino Acid Content in Onions as Potential Fingerprints of Geographical Origin: The Case of Rossa da Inverno sel. Rojo Duro.

In this study, we were interested in comparing the amino acid profile in a specific variety of onion, Rossa da inverno sel. Rojo Duro, produced in two different Italian sites: the Cannara (Umbria region) and Imola (Emilia Romagna region) sites. Onions were cultivated in a comparable manner, mostly in terms of the mineral fertilization, seeding, and harvesting stages, as well as good weed control. Furthermore, in both regions, the plants were irrigated by the water sprinkler method and subjected to similar temperature and weather conditions. A further group of Cannara onions that were grown by micro-irrigation was also evaluated. After the extraction of the free amino acid mixture, an ion-pairing reversed-phase (IP-RP) HPLC method allowed for the separation and the evaporative light scattering detection of almost all the standard proteinogenic amino acids. However, only the peaks corresponding to leucine (Leu), phenylalanine (Phe), and tryptophan (Trp), were present in all the investigated samples and they were unaffected from the matrix interfering peaks. The use of the beeswarm/box plots revealed that the content of Leu and Phe were markedly influenced by the geographical origin of the onions (with *** p.

Chiral HPLC Separation and Modeling of Four Stereomers of DL-Leucine-DL-Tryptophan Dipeptide on Amylose Chiral Column.

Chiral high-performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL-leucine-tryptophan DL-dipeptide on AmyCoat-RP column are described. The mobile phase applied was ammonium acetate (10 mM)-methanol-acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL-, DD-, DL-, and LD- stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0-2.3 and 5.6-14.0 µg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π-π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL-leucine-DL-tryptophan in unknown matrices. Chirality 28:642-648, 2016. © 2016 Wiley Periodicals, Inc.

6-bromohypaphorine from marine nudibranch mollusk Hermissenda crassicornis is an agonist of human α7 nicotinic acetylcholine receptor.

6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4ß2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 µM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 µM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes.

[Tryptophan immunoadsorption for multiple sclerosis and neuromyelitis optica: therapy option for acute relapses during pregnancy and breastfeeding]. / Tryptophan-Immunadsorption bei Multipler Sklerose und Neuromyelitis optica : Therapieoption bei akuten Schüben in der Schwangerschaft und Stillphase.

BACKGROUND: Approximately 25 % of women with multiple sclerosis (MS) suffer clinically relevant relapses during pregnancy. Almost all disease-modifying drugs are contraindicated in pregnancy. High-dose glucocorticoids have some serious risks, especially within the first trimester. Tryptophan immunoadsorption (IA) provides a safe option to treat MS relapses during pregnancy. OBJECTIVES: In this case series we describe for the first time the use of tryptophan IA for MS and neuromyelitis optica (NMO) relapses during pregnancy and breastfeeding. PATIENTS AND METHODS: In this study a total of 9 patients were retrospectively analyzed of which 7 patients received IA treatment during pregnancy, 2 during breastfeeding and 4-6 tryptophan IA treatments were performed per patient with the single use tryptophan adsorber. Primary outcome was symptom improvement of the relapse. RESULTS: In this study four patients with MS and one with NMO relapse during pregnancy were treated with IA without preceding glucocorticoid pulse therapy. The MS patients showed improvement in the expanded disability status scale (EDSS) by at least one point, the NMO patient showed significant improvement in visual acuity and two pregnant patients with steroid-refractory relapses showed clinically relevant improvement after IA. Of the patients two suffered from steroid-refractory relapses during breastfeeding and relapse symptoms improved in both cases after treatment with IA. All treatments were well tolerated and no serious adverse events occurred. CONCLUSION: Tryptophan IA was found to be safe, well-tolerated and effective in the treatment of MS and NMO relapses during pregnancy and breastfeeding, sometimes without preceding glucocorticoid pulse therapy. A binding recommendation is limited without prospective clinical studies.

Enantiomeric separation by microchip electrophoresis using bovine serum albumin conjugated magnetic core-shell Fe3 O4 @Au nanocomposites as stationary phase.

In this work, a novel enantioselective MCE was developed employing BSA-conjugated Fe3 O4 @Au nanoparticles (Fe3 O4 @Au NPs) as stationary phase. Fe3 O4 @Au NPs with high magnetic responsively, excellent solubility, and high dispersibility in water were prepared through a sonochemical synthesis strategy. BSA was then immobilized onto the Fe3 O4 @Au NPs surfaces through the well-developed interaction between Au NPs and amine groups of BSA to form Fe3 O4 @Au NPs-BSA conjugates, which were then locally packed into PDMS microchannels with the help of magnets. The resultant Fe3 O4 @Au NPs-BSA conjugates not only have the magnetism of Fe3 O4 NPs that make them easily manipulated by an external magnetic field, but also have the larger surface and excellent biocompatibility of Au shell, which can incorporate much more biomolecules and well maintain their biological activity. In addition, the successful BSA decorations endowed Fe3 O4 @Au NPs-BSA conjugates with pH-tunable water solubility related to the pI of BSA (pI 4.7) and led to enhanced stability against high ionic strength. Compared with the native PDMS microchannel, the modified surfaces exhibited more stable and suppressed electroosmotic mobility, and less nonspecific adsorption toward analytes. Successful separation of chiral amino acids (tryptophan and threonine) and ofloxacin enantiomers demonstrate that the constructed MCE columns own ideal enantioselectivity. The results are expected to open up a new possibility for high-throughput screening of enantiomers with protein targets as well as a new application of magnetic NPs.

Isolation, structure determination and cytotoxicity studies of tryptophan alkaloids from an Australian marine sponge Hyrtios sp.

Mass-guided fractionation of the MeOH extract from a specimen of the Australian marine sponge Hyrtios sp. resulted in the isolation of two new tryptophan alkaloids, 6-oxofascaplysin (2), and secofascaplysic acid (3), in addition to the known metabolites fascaplysin (1) and reticulatate (4). The structures of all molecules were determined following NMR and MS data analysis. Structural ambiguities in 2 were addressed through comparison of experimental and DFT-generated theoretical NMR spectral values. Compounds 1-4 were evaluated for their cytotoxicity against a prostate cancer cell line (LNCaP) and were shown to display IC50 values ranging from 0.54 to 44.9 µM.

The pivotal role of copper(II) in the enantiorecognition of tryptophan and histidine by gold nanoparticles.

Stereoselective amino acid analysis has increasingly moved into the scope of interest of the scientific community. In this work, we report a study on the chiral recognition of D,L-Trp and D,L-His using L -Cys-capped gold nanoparticles (AuNPs) and copper(II) ion. In the L -Cys-capped AuNPs, the thiol group of the amino acid interacts with AuNPs through the formation of Au­S bond, whereas the α-amino and α-carboxyl groups of the surface-confined cysteine can coordinate the copper(II) ion, which in turn, binds the L- or D-amino acid present in solution forming diastereoisomeric complexes. The resulting systems have been characterized by UV­Vis spectra and dynamic light scattering measurements, obtaining different results for L- and D-Trp, as well as for L- and D-His. The knowledge of the solution equilibria of the investigated systems allowed us to accurately calculate in advance the concentrations of the species presentin solution and to optimize the system performances, highlighting the pivotal role of copper(II) ion in the enantiodiscrimination processes.

Separation of tryptophan enantiomers by ligand-exchange chromatography with novel chiral ionic liquids ligand.

Chiral ionic liquids (CILs) with amino acids as cations have been applied as novel chiral ligands coordinated with Cu(2+) to separate tryptophan enantiomers in ligand exchange chromatography. Four kinds of amino acid ionic liquids, including [L-Pro][CF3COO], [L-Pro][NO3], [L-Pro]2[SO4], and [L-Phe][CF3COO] were successfully synthesized and used for separation of tryptophan enantiomers. To optimize the separation conditions, [L-Pro][CF3COO] was selected as the model ligand. Some factors influencing the efficiency of chiral separation, such as copper ion concentration, CILs concentration, methanol ratio (methanol/H2O, v/v), and pH, were investigated. The obtained optimal separation conditions were as follows: 8.0 mmol/L Cu(OAc)2, 4.0 mmol/L [L-Pro][CF3COO], and 20% (v/v) methanol at pH 3.6. Under the optimum conditions, acceptable enantioseparation of tryptophan enantiomers could be observed with a resolution of 1.89. The results demonstrate the good applicability of CILs with amino acids as cations for chiral separation. Furthermore, a comparative study was also conducted for exploring the mechanism of the CILs as new ligands in ligand exchange chromatography.

Chiral separation of phenylalanine and tryptophan by capillary electrophoresis using a mixture of ß-CD and chiral ionic liquid ([TBA] [L-ASP]) as selectors.

In this paper, a simple, effective and green capillary electrophoresis separation and detection method was developed for the quantification of underivatized amino acids (dl-phenylalanine; dl-tryptophan) using ß-Cyclodextrin and chiral ionic liquid ([TBA] [l-ASP]) as selectors. Separation parameters such as buffer concentrations, pH, ß-CD and chiral ionic liquid concentrations and separation voltage were investigated for the enantioseparation in order to achieve the maximum possible resolution. A good separation was achieved in a background electrolyte composed of 15 mm sodium tetraborate, 5 mm ß-CD and 4 mm chiral ionic liquid at pH 9.5, and an applied voltage of 10 kV. Under optimum conditions, linearity was achieved within concentration ranges from 0.08 to 10 µg/mL for the analytes with correlation coefficients from 0.9956 to 0.9998, and the analytes were separated in less than 6 min with efficiencies up to 970,000 plates/m. The proposed method was successfully applied to the determination of amino acid enantiomers in compound amino acids injections, such as 18AA-I, 18AA-II and 3AA.

Preparation and characterization of a polystyrene/bovine serum albumin nanoparticle-coated capillary for chiral separation using open-tubular capillary electrochromatography.

Polystyrene (PS) nanoparticles coated by BSA, hereafter denoted as PS/BSA, were prepared and chemically immobilized for the first time onto a capillary inner wall for open-tubular CEC (OTCEC). EOF and scanning electron micrography were used to characterize the prepared nanoparticle-coated capillaries. To investigate the performance of the prepared columns in OTCEC, chiral separation of d,l-tryptophan (dl-Trp) was performed in monolayer BSA-modified capillary and PS/BSA nanoparticle-coated columns. The results indicated that the nanoparticle-modified column afforded a higher resolution compared with the monolayer type. Rapid enantioseparation of dl-Trp (within 3 min) was achieved with the PS/BSA-immobilized column using an electroosmotic pump-assisted CEC. Enantiomer separations of other compounds like dl-tyrosine and warfarin were also achieved with the column. Besides, run-to-run and column-to-column repeatabilities of the PS/BSA-coated column in the chiral separation were systematically introduced.

Preparative separation of enantiomers based on functional nucleic acids modified gold nanoparticles.

The preparative-scale separation of chiral compounds is vitally important for the pharmaceutical industry and related fields. Herein we report a simple approach for rapid preparative separation of enantiomers using functional nucleic acids modified gold nanoparticles (AuNPs). The separation of DL-tryptophan (DL-Trp) is demonstrated as an example to show the feasibility of the approach. AuNPs modified with enantioselective aptamers were added into a racemic mixture of DL -Trp. The aptamer-specific enantiomer (L-Trp) binds to the AuNPs surface through aptamer-L-Trp interaction. The separation of DL-Trp is then simply accomplished by centrifugation: the precipitate containing L-Trp bounded AuNPs is separated from the solution, while the D-Trp remains in the supernatant. The precipitate is then redispersed in water. The aptamer is denatured under 95 °C and a second centrifugation is then performed, resulting in the separation of AuNPs and L-Trp. The supernatant is finally collected to obtain pure L-Trp in water. The results show that the racemic mixture of DL-Trp is completely separated into D-Trp and L-Trp, respectively, after 5 rounds of repeated addition of fresh aptamer-modified AuNPs to the DL-Trp mixture solution. Additionally, the aptamer-modified AuNPs can be repeatedly used for at least eight times without significant loss of its binding ability because the aptamer can be easily denatured and renatured in relatively mild conditions. The proposed approach could be scaled up and extended to the separation of other enantiomers by the adoption of other enantioselective aptamers.

Structural characterization of new microcystins containing tryptophan and oxidized tryptophan residues.

Microcystins are cyclic peptides produced by cyanobacteria, which can be harmful to humans and animals when ingested. Eight of the (more than) 90 microcystin variants presently characterized, contain the amino acid tryptophan. The well-researched oxidation products of tryptophan; kynurenine, oxindolylalanine, and N-formylkynurenine, have been previously identified in intact polypeptides but microcystin congeners containing oxidized tryptophan moieties have not been reported. Liquid chromatography-tandem mass spectrometric analysis of an extract of Microcystis CAWBG11 led to the tentative identification of two new tryptophan-containing microcystins (MC­WAba and MC-WL), as well as eight other microcystin analogs containing kynurenine, oxindolylalanine and N­formylkynurenine (Nfk). Investigation of one of these congeners (MC­NfkA) by nuclear magnetic resonance spectroscopy was used to verify the presence of Nfk in the microcystin. Liquid chromatography-mass spectrometry analysis of a tryptophan oxidation experiment demonstrated that tryptophan-containing microcystins could be converted into oxidized tryptophan analogs and that low levels of oxidized tryptophan congeners were present intracellularly in CAWBG11. MC-NfkR and MC-LNfk were detected in standards of MC-WR and MC-LW, indicating that care during storage of tryptophan-containing microcystins is required.

UPLC-PDA-ESI-MS/MS analysis of compounds extracted by cardiac h9c2 cell from Polygonum orientale.

INTRODUCTION: 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. OBJECTIVE: 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. METHODOLOGY: 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. RESULTS: 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. CONCLUSION: 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.