共查询到20条相似文献,搜索用时 31 毫秒
1.
Qian Zhang Kyle A. Noble Yuan Mao Nicolas L. Young Shridhar K. Sathe Kenneth H. Roux Alan G. Marshall 《Journal of the American Society for Mass Spectrometry》2013,24(7):1016-1025
The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model. Figure
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2.
Stephen J. Coales Steven J. Tuske Justine C. Tomasso Yoshitomo Hamuro 《Rapid communications in mass spectrometry : RCM》2009,23(5):639-647
The epitope of horse cytochrome c against monoclonal antibody E8 was determined using amide hydrogen/deuterium (H/D) exchange combined with immobilized antibody, on‐line pepsin proteolysis, liquid chromatography (LC), and mass spectrometry (MS). The results were generally in good agreement with contact residues identified by an X‐ray co‐crystal structure of the E8–cytochrome c complex and results obtained by H/D exchange with nuclear magnetic resonance (NMR) spectrometry. The H/D exchange reaction of cytochrome c was carried out in the presence or absence of immobilized E8 antibody. Regions that gained less deuterium in the presence of the antibody than in its absence are defined as the epitope by the H/D exchange MS method. Control experiments were carefully designed to help identify the epitope with high confidence. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
3.
Tatiana Pimenova Lukas Meier Bernd Roschitzki Gabriela Paraschiv Michael Przybylski Renato Zenobi 《Analytical and bioanalytical chemistry》2009,395(5):1395-1401
The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and
treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be
effectively identified by the proteolytic epitope excision–mass spectrometry (MS) method, which involves (1) immobilization
of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of
the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s).
In the epitope analysis of recombinant cellular bovine prion protein (bPrPC) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding
of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde
functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision–MS, with complete
suppression of nonspecific bPrP binding. 相似文献
4.
《Electrophoresis》2018,39(16):2069-2082
High‐resolution capillary zone electrophoresis – mass spectrometry (CZE‐MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle‐down and intact CZE‐MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post‐translational modifications (PTMs) and glycosylation structures. Middle‐down and intact CZE separations were performed in an acidified methanol‐water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle‐down analysis of the IdeS‐digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X‐deamidated, 1X‐deamidated, and non‐deamidated forms at baseline resolution. In the course of the middle‐down CZE‐MS analysis, separation of glycoforms of the FC/2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE‐MS2. Incorporation of TCEP‐HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE‐MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X‐glycosylated, 1X‐glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE‐MS represents a complementary approach to the more conventional liquid‐chromatography – mass spectrometry‐based approaches. 相似文献
5.
Two new 3,4‐seco‐cycloartane triterpenes, named sootepin F ( 1 ) and sootepin G ( 2 ), together with two known compounds, coronalolide methyl ester ( 3 ) and sootepin D ( 4 ), were isolated from the leaves and twigs of Gardenia sootepensis. Their structures were elucidated on the basis of 1D‐ and 2D‐NMR experiments, including HMBC, HSQC, 1H,1H‐COSY, and ROESY, as well as HR‐MS. 相似文献
6.
Dalila Smati Anne‐Claire Mitaine‐Offer Tomofumi Miyamoto V. Hammiche Marie‐Aleth Lacaille‐Dubois 《Helvetica chimica acta》2007,90(4):712-719
Four new ursane‐based triterpene glycosides, compounds 1 – 4 , as well as the known glycosides zygophylosides E, G, and H, and 3‐O‐(β‐D ‐quinovopyranosyl)quinovic acid 28‐(O‐β‐D ‐glucopyranosyl) ester, were isolated from the BuOH‐soluble fraction of the MeOH/H2O 7 : 3 extracts of Zygophyllum geslini (roots or aerial parts). Their structures were established mainly by 1D‐ and 2D‐NMR techniques, in combination with HR‐MS analysis and acid hydrolysis. 相似文献
7.
The unusual hydrogen‐deuterium exchange of α‐carbon protons in N‐substituted glycine‐containing peptides 下载免费PDF全文
Remigiusz Bąchor Bartosz Setner Alicja Kluczyk Piotr Stefanowicz Zbigniew Szewczuk 《Journal of mass spectrometry : JMS》2014,49(1):43-49
Hydrogens connected to α‐carbon (α‐C) of amino acid residues are usually resistant to hydrogen‐deuterium exchange (HDX) unless reaction conditions promote racemization. Although N‐methylglycine (sarcosine) residue has been found in biologically active peptide such as cyclosporine, to the best of our knowledge, the HDX of α‐C protons of this residue was not explored yet. Here, we presented a new and efficient methodology of α‐C deuteration in sarcosine residues under basic aqueous conditions. The deuterons, introduced at α‐C atom, do not undergo back‐exchange in acidic aqueous solution. The electrospray ionization‐MS and MS/MS experiments on proposed model peptides confirmed the HDX at α‐C and revealed the unexpected hydrogen scrambling in sarcosine‐containing peptides. Although the observed HDX of α‐C protons is only successful in N‐acylglycine when the amide possesses a certain degree of alkylation, it offers a new approach to the analysis of sarcosine‐containing peptides such as cyclosporine. Copyright © 2014 John Wiley & Sons, Ltd. 相似文献
8.
Elise Pinta Katarzyna A. Duda Dr. Anna Hanuszkiewicz Dr. Zbigniew Kaczyński Dr. Buko Lindner Dr. Wayne L. Miller Dr. Heidi Hyytiäinen Dr. Christian Vogel Dr. Sabine Borowski Katarzyna Kasperkiewicz Dr. Joseph S. Lam Dr. Joanna Radziejewska‐Lebrecht Dr. Mikael Skurnik Dr. Otto Holst Dr. 《Chemistry (Weinheim an der Bergstrasse, Germany)》2009,15(38):9747-9754
The outer core (OC) region of Yersinia enterocolitica serotype O:3 lipopolysaccharide is a hexasaccharide essential for the integrity of the outer membrane. It is involved in resistance against cationic antimicrobial peptides and plays a role in virulence during early phases of infection. We show here that the proximal residue of the OC hexasaccharide is a rarely encountered 4‐keto‐hexosamine, 2‐acetamido‐2,6‐dideoxy‐D ‐xylo‐hex‐4‐ulopyranose (Sugp) and that WbcP is a UDP‐GlcNAc‐4,6‐dehydratase enzyme responsible for the biosynthesis of the nucleotide‐activated form of this rare sugar converting UDP‐2‐acetamido‐2‐deoxy‐D ‐glucopyranose (UDP‐D ‐GlcpNAc) to UDP‐2‐acetamido‐2,6‐dideoxy‐D ‐xylo‐hex‐4‐ulopyranose (UDP‐ Sugp). In an aqueous environment, the 4‐keto group of this sugar was present in the 4‐dihydroxy form, due to hydration. Furthermore, evidence is provided that the axial 4‐hydroxy group of this dihydroxy function was crucial for the biological role of the OC, that is, in the bacteriophage and enterocoliticin receptor structure and in the epitope of a monoclonal antibody. 相似文献
9.
Delineation of G‐Quadruplex Alkylation Sites Mediated by 3,6‐Bis(1‐methyl‐4‐vinylpyridinium iodide)carbazole‐Aniline Mustard Conjugates 下载免费PDF全文
Chien‐Han Chen Tsung‐Hao Hu Tzu‐Chiao Huang Ying‐Lan Chen Prof. Yet‐Ran Chen Prof. Chien‐Chung Cheng Prof. Chao‐Tsen Chen 《Chemistry (Weinheim an der Bergstrasse, Germany)》2015,21(48):17379-17390
A new G‐quadruplex (G‐4)‐directing alkylating agent BMVC‐C3M was designed and synthesized to integrate 3,6‐bis(1‐methyl‐4‐vinylpyridinium iodide)carbazole (BMVC) with aniline mustard. Various telomeric G‐4 structures (hybrid‐2 type and antiparallel) and an oncogene promoter, c‐MYC (parallel), were constructed to react with BMVC‐C3M, yielding 35 % alkylation yield toward G‐4 DNA over other DNA categories (<6 %) and high specificity under competition conditions. Analysis of the intact alkylation adducts by electrospray ionization mass spectroscopy (ESI‐MS) revealed the stepwise DNA alkylation mechanism of aniline mustard for the first time. Furthermore, the monoalkylation sites and intrastrand cross‐linking sites were determined and found to be dependent on G‐4 topology based on the results of footprinting analysis in combination with mass spectroscopic techniques and in silico modeling. The results indicated that BMVC‐C3M preferentially alkylated at A15 (H26), G12 (H24), and G2 (c‐MYC), respectively, as monoalkylated adducts and formed A15–C3M–A21 (H26), G12–C3M–G4 (H24), and G2–C3M–G4/G17 (c‐MYC), respectively, as cross‐linked dialkylated adducts. Collectively, the stability and site‐selective cross‐linking capacity of BMVC‐C3M provides a credible tool for the structural and functional characterization of G‐4 DNAs in biological systems. 相似文献
10.
Anil Khanal Yan Pan Leonid S. Brown Lars Konermann 《Journal of mass spectrometry : JMS》2012,47(12):1620-1626
Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) are a well‐established tool for water‐soluble proteins. To the best of our knowledge, the current study is the first that applies this approach to an integral membrane protein. The native state of bacteriorhodopsin (BR) comprises seven transmembrane helices and a covalently bound retinal cofactor. BR exposure to sodium dodecyl sulfate (SDS) induces partial unfolding and retinal loss. We employ a custom‐built three‐stage mixing device for pulsed‐HDX/MS investigations of BR refolding. The reaction is triggered by mixing SDS‐denatured protein with bicelles. After a variable folding time (10 ms to 24 h), the protein is exposed to excess D2O buffer under rapid exchange conditions. The HDX pulse is terminated by acid quenching after 24 ms. Subsequent off‐line analysis is performed by size exclusion chromatography and electrospray MS. These measurements yield the number of protected backbone N–H sites as a function of folding time, reflecting the recovery of secondary structure. Our results indicate that much of the BR secondary structure is formed quite late during the reaction, on a time scale of 10 s and beyond. It is hoped that in the future it will be possible to extend the pulsed‐HDX/MS approach employed here to membrane proteins other than BR. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献
11.
《Electroanalysis》2018,30(3):402-414
A sensitive electrochemical immunosensor for Hepatitis B virus surface antigen (HBsAg) detection was fabricated based on hemin/G‐quadruplex interlaced onto Fe3O4‐AuNPs or hemin ‐amino‐reduced graphene oxide nanocomposite (H‐amino‐rGO‐Au). G‐quadruplex DNAzyme, which is composed of hemin and guanine‐rich nucleic acid, is an effective signal amplified tool for its outstanding peroxidase activity and Fe3O4‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites with quasi‐enzyme activity provide appropriate support for the immobilization of hemin/G‐quadruplex. The target protein was sandwiched between the primary antibody immobilized on the GO and secondary antibody immobilized on the Fe3O4‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites and glutaraldehyde was used as linking agent for the immobilization of primary antibody on the surface of GO. Both Fe3O4‐AuNPs and H‐amino‐rGO‐Au nanocomposite and also hemin/G‐quadruplex can cooperate the electrocatalytic reduction of H2O2 in the presence of methylene blue as mediator. The proposed immunosensor has a wide linear dynamic range of 0.1 pg/ml to 300 pg/ml with a detection limit of 60 fg/ml when Fe3O4‐AuNPs was used for immobilization of hemin/G‐quadruplex, while the dynamic range and DL were 0. 1–1000 pg/mL and 10 fg/mL, respectively in the presence of H‐amino‐rGO‐ Au nanocomposite as platform for immobilizing of hemin/G‐quadruplex. The proposed immunosensor was also used for analysis of HBsAg in spiked human serum samples with satisfactory results. 相似文献
12.
Ganesh R. Jadhav Mohammad U. Shaikh Murlidhar S. Shingare Charansingh H. Gill 《Journal of heterocyclic chemistry》2008,45(5):1287-1291
Synthesis of some novel 2‐{2‐[1‐(3‐substitutedphenyl)‐1H‐1,2, 3‐triazol‐4‐yl‐]ethyl)‐1H‐benzo[d]‐imidazole derivatives, by the condensation of o‐phenylenediamine with 3‐(1‐(3‐substituted‐phenyl)‐1H‐1,2,3‐triazol‐4‐yl) propanoic acid and then subsequent reactions with different substituted alkyl halides as electrophiles are mentioned. The synthesized compounds were characterized by 1H NMR, EI‐MS and IR spectroscopic techniques. 相似文献
13.
Faiza Nessark Mohamed Eissa Abdoullatif Baraket Nadia Zine Belkacem Nessark Ahmed Zouaoui Joan Bausells Abdelhamid Errachid 《Electroanalysis》2020,32(8):1795-1806
In the present study, we developed a novel label‐free capacitance impedimetric immunosensor based on the immobilization of the human monoclonal antibody anti‐interleukin‐10 (anti‐IL‐10 mAb) onto polypyrrole (PPy)‐modified silicon nitride (Si3N4) substrates. The immunosensor was used for the detection of the recombinant interleukin‐10 antigen (rh IL‐10) that may be secreted in patients at the early stage of inflammation. The immunosensor was created by chemical deposition of PPy conducting layer on pyrrole?silane (SPy)‐treated Si/SiO2/Si3N4 substrates (Si/SiO2/Si3N4?SPy), followed by anti‐IL‐10 mAb immobilization through carboxyl‐functionalized diazonium (CMA) protocol and carbodiimide chemistry. The surface characterization and the biofunctionalization steps were characterized by SEM, FTIR and cyclic voltammetry (CV) while the detection process was carried out by using electrochemical impedance spectroscopy (EIS) analyses. The created immunosensor showed two linear fittings (R2=0.999) for the detection of rh IL‐10 within the concentration range from 1–50 pg/mL. It exhibited high sensitivity (0.1128 (pg/mL)?1) with a very low limit of detection (LOD)=0.347 pg/mL, more particularly, at the low concentration range (1–10 pg/mL). Thus, this developed polypyrrole‐based immunosensor represents a promising strategy for creation of miniaturized label‐free, fast and highly sensitive biosensors for diagnosis of inflammation biomarkers at very low concentrations with reduced cost. 相似文献
14.
Stefanescu R Born R Moise A Ernst B Przybylski M 《Journal of the American Society for Mass Spectrometry》2011,22(1):148-157
Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor
is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically
to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification
of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution
MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen
was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination
and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a
Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to
the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized
on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound
peptides provided the identification of two specific epitope peptides (5–16) and (17–23) that showed high affinity to the
antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody. 相似文献
15.
Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability 下载免费PDF全文
Dr. Matthew J. Edgeworth Dr. Jonathan J. Phillips Dr. David C. Lowe Dr. Alistair D. Kippen Dr. Daniel R. Higazi Prof. James H. Scrivens 《Angewandte Chemie (International ed. in English)》2015,54(50):15156-15159
Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion‐mobility mass spectrometry (IM‐MS) and hydrogen/deuterium exchange mass spectrometry (HDX‐MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins. 相似文献
16.
A monoclonal antibody (mAb) to P‐glycoprotein (Pgp), UIC2, is used as a targeting moiety for N‐(2‐hydroxypropyl)methacrylamide (HPMA) copolymer/drug [(meso chlorin e6 mono(N‐2‐aminoethylamide) (Mce6) or doxorubicin (DOX)] conjugates to investigate their cytotoxicity towards the Pgp‐expressing human ovarian carcinoma cell line A2780/AD. The binding, internalization, and subcellular trafficking of a fluorescein labeled UIC2 targeted HPMA copolymer are studied and show localization to the plasma membrane with limited internalization. The specificity of the UIC2‐targeted HPMA copolymer/drug conjugates are confirmed using the sensitive cell line A2780 that does not express Pgp.
17.
Tingting Tu Mihaela Drăgu§anu Brînduşa-Alina Petre Don L. Rempel Michael Przybylski Michael L. Gross 《Journal of the American Society for Mass Spectrometry》2010,21(10):1660-1667
A new methodology using hydrogen/deuterium amide exchange (HDX) to determine the binding affinity of protein-peptide interactions
is reported. The method, based on our previously established approach, protein ligand interaction by mass spectrometry, titration,
and H/D exchange (PLIMSTEX) [J. Am. Chem. Soc.
2003, 125, 5252–5253], makes use of a dilution strategy (dPLIMSTEX) for HDX, using the mass of the peptide ligand as readout. We employed
dPLIMSTEX to study the interaction of calcium-saturated calmodulin with the opioid peptide β-endorphin as a model system;
the affinity results are in good agreement with those from traditional PLIMSTEX and with literature values obtained by using
other methods. We show that the dPLIMSTEX method is feasible to quantify an antigen-antibody interaction involving a 3-nitrotyrosine
modified peptide in complex with a monoclonal anti-nitrotyrosine antibody. A dissociation constant in the low nanomolar range
was determined, and a binding stoichiometry of antibody/peptide of 1:2 was confirmed. In addition, we determined that the
epitope in the binding interface contains a minimum of five amino acids. The dPLIMSTEX approach is a sensitive and powerful
tool for the quantitative determination of peptide affinities with antibodies, complementary to conventional immuno-analytical
techniques. 相似文献
18.
Simultaneous determination of morphine‐6‐d‐glucuronide,morphine‐3‐d‐glucuronide and morphine in human plasma and urine by ultra‐performance liquid chromatography–tandem mass spectrometry: Application to M6G injection pharmacokinetic study 下载免费PDF全文
《Biomedical chromatography : BMC》2018,32(2)
A robust ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the determination of morphine‐6‐d ‐glucuronide (M6G), morphine‐3‐d ‐glucuronide (M3G) and morphine (MOR) in human plasma and urine has been developed and validated. The analytes of interest were extracted from plasma by protein precipitation. The urine sample was prepared by dilution. Both plasma and urine samples were chromatographed on an Acquity UPLC HSS T3 column using gradient elution. Detection was performed on a Xevo TQ‐S tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. Matrix interferences were not observed at the retention time of the analytes and internal standard, naloxone‐D5. The lower limits of quantitation of plasma and urine were 2/0.5/0.5 and 20/4/2 ng/mL for M6G/M3G/MOR, respectively. Calibration curves were linear over the concentration ranges of 2–2000/0.5–500/0.5–500 and 20–20,000/4–4000/2–2000 ng/mL for M6G/M3G/MOR in plasma and urine samples, respectively. The precision was <7.14% and the accuracy was within 85–115%. Furthermore, stability of the analytes at various conditions, dilution integrity, extraction recovery and matrix effect were assessed. Finally, this quantitative method was successfully applied to the pharmacokinetic study of M6G injection in Chinese noncancer pain patients. 相似文献
19.
Bis‐C‐pivot macrocycles containing aminophosphonate functions ( 5–10 ) have been synthesized and characterized by elemental analysis, FTIR, MS, 1D 1H, 13C and 31P NMR, and 2D HETCOR techniques. The phosphorylation reaction of dibenzo‐bis‐imino crown ethers ( 1–4 ) with dimethyl and diethyl phosphite used here has the potential to provide bis‐C‐pivot macrocycles ( 5–10 ), which possess two stereogenic C‐centers giving rise to diastereoisomers (meso and racemic). Detailed spectral assignments for the meso and racemic forms of the compounds are reported on the basis of chemical shifts, signal intensities, spin–spin coupling constants, and splitting patterns. The bis‐C‐pivot macrocycles ( 5–10 ) may serve as a potential new class of supramolecular host molecules. 相似文献
20.
Wen Su Qi Liu Qing Yang Jingang Yu Xiaoqing Chen 《Journal of separation science》2013,36(20):3338-3344
An off‐line 2D high‐speed counter‐current chromatography technique in preparative scale has been successfully applied to separate and purify the main compounds from the ethyl acetate extract of Desmodium styracifolium. A two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water at an optimized volume ratio of 1:2:1:2 v/v/v/v was used. Conventional high‐speed counter‐current chromatography was used as the first dimension, and the upper phase of the solvent system was used as the stationary phase in the head‐to‐tail elution mode at a flow rate of 2.0 mL/min and a rotation speed of 900 rpm. Recycling high‐speed counter‐current chromatography served as the second dimension to separate an impure fraction of the first dimension. A total of four well‐separated substances including vanillic acid ( 1 ), β‐sitosterol ( 2 ), formononetin ( 3 ), and aromadendrin ( 4 ) were obtained, and their purities and structures were identified by HPLC–MS and 1H NMR spectroscopy. The results illustrated that off‐line 2D high‐speed counter‐current chromatography is an effective way to isolate compounds in complex samples. 相似文献