Screening anti‐allergic components of Astragali Radix using LAD2 cell membrane chromatography coupled online with UHPLC‐ESI‐MS/MS method

Huangqi (Astragali Radix), a traditional Chinese herb, is widely used in clinical therapy in China. In addition, an anti‐allergic effect of constituents in Huangqi has been reported in the scientific literature. In the present study, cell membrane chromatography coupled online with UHPLC‐ESI‐MS/MS method was developed to screen, analyze and identify the anti‐allergic components of Huangqi. The Laboratory of Allergic Disease 2 (LAD2) cell was used to establish cell membrane chromatography, which was combined with UHPLC‐ESI‐MS/MS. The coupled system was then used to screen anti‐allergic components from Huangqi. Effects of active components were verified by histamine release assay. A component retained on the LAD2 cell membrane chromatography was identified as formononetin. Bioactivity of formononetin was investigated by histamine release assay in LAD2 cells, and it was found that formononetin could inhibit histamine release in a dose‐dependent manner from 1 to 100 μm . The LAD2 cell membrane chromatography online with UHPLC‐ESI‐MS/MS method is an effective technique for screening the anti‐allergic components of Huangqi.     

Screening anaphylactic components of MaiLuoNing injection by using rat basophilic leukemia‐2H3 cell membrane chromatography coupled with HPLC–ESI‐TOF‐MS

MaiLuoNing injection is a traditional Chinese medicine that used clinically since the 1950s in China. However, anaphylactic reactions, through the potentiation of mast cell degranulation, have been reported. In the present study, a rat basophilic leukemia‐2H3 cell membrane chromatography coupled with high‐performance liquid chromatography and electrospray ionization‐ion trap‐time of flight‐mass spectrometry method was established for screening, analyzing, and identifying the potential anaphylactic components of MaiLuoNing injection. Harpagoside, a potential degranulator of rat basophilic leukemia‐2H3 cells, was retained in rat basophilic leukemia‐2H3 cell membrane chromatography. We aimed to evaluate the retained components to determine which of those were capable of inducing degranulation of basophilic leukemia cells. A β‐hexosaminidase assay revealed that harpagoside can induce rat basophilic leukemia‐2H3 cell degranulation in a dose‐dependent manner. BLBA/c mice also exhibit passive cutaneous anaphylaxis in response to harpagoside. These results indicate that rat basophilic leukemia‐2H3 cell membrane chromatography coupled with high‐performance liquid chromatography and electrospray ionization ion trap time‐of‐flight mass spectrometry is effective in screening for the anaphylactic components of MaiLuoNing injection.     

Cell membrane chromatography coupled online with LC‐MS to screen anti‐anaphylactoid components from Magnolia biondii Pamp. targeting on Mas‐related G protein‐coupled receptor X2

Mas‐related G protein‐coupled receptor X2 was a mast cell–specific receptor mediating anaphylactoid reactions by activating mast cells degranulation, and it was also identified as a target for modulating mast cell–mediated anaphylactoid and inflammatory diseases. The anti‐anaphylactoid drugs used clinically disturb the partial effect of partial mediators released by mast cells. The small molecule of Mas‐related G protein‐coupled receptor X2 specific antagonists may provide therapeutic action for the anaphylactoid and inflammatory diseases in the early stage. In this study, the Mas‐related G protein‐coupled receptor X2 high expression cell membrane chromatography was coupled online with liquid chromatography and mass spectrometry and successfully used to screen anti‐anaphylactoid components from Magnolia biondii Pamp. Fargesin and pinoresinol dimethyl ether were identified as potential anti‐anaphylactoid components. Bioactivity of these two components were investigated by β hexosaminidase and histamine release assays on mast cells, and it was found that these two components could inhibit β hexosaminidase and histamine release in a concentration‐dependent manner. This Mas‐related G protein‐coupled receptor X2 high expression cell membrane chromatography coupled online with liquid chromatography and mass spectrometry system could be applied for screening potential anti‐anaphylactoid components from natural medicinal herbs. This study also provided a powerful system for drug discovery in natural medicinal herbs.     

Identification of ilaprazole metabolites in human urine by HPLC‐ESI‐MS/MS and HPLC‐NMR experiments

Ilaprazole is a new proton pump inhibitor designed for the treatment of gastric ulcers, and limited data is available on the metabolism of the drug. In this article, the structural elucidation of urinary metabolites of ilaprazole in human was described by HPLC‐ESI‐MS/MS and stopped‐flow HPLC‐NMR experiments. Urinary samples were precipitated by sodium carbonate solution, and then extracted by liquid–liquid extraction after adding ammonium acetate buffer solution. The enriched sample was separated using a C₁₈ reversed‐phase column with the mobile phase composed of acetonitrile and 0.05 mol/L ammonium acetate buffer solution in a gradient solution, and then directly coupled to ESI‐MS/MS detection in an on‐line mode or ¹H‐NMR (500 MHz) spectroscopic detection in a stopped‐flow mode. As a result, four sulfide metabolites, ilaprazole sulfide (M1), 12‐hydroxy‐ilaprazole sulfide (M2), 11,12‐dihydroxy‐ilaprazole sulfide (M3) and ilaprazole sulfide A (M4), were identified by comparing their MS/MS and NMR data with those of the parent drug and available standard compounds. The main biotransformation reactions of ilaprazole were reduction and the aromatic hydroxylation of the parent drug and its relative metabolites. The result testified that HPLC‐ESI‐MS/MS and HPLC‐NMR could be widely applied in detection and identification of novel metabolites. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on‐line two‐dimensional reversed‐phase liquid chromatography/electrospray ionization–tandem mass spectrometry (2D‐LC‐ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D‐LC‐ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C₁₈ column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5–10 ng/mL (r² > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of hydroxyl radical‐induced oxidation process of glucagon by reversed‐phase HPLC and ESI‐MS/MS

Structural modification of a polypeptide hormone, glucagon, by a hydroxyl radical in vitro was investigated by reversed‐phase high‐performance liquid chromatography (RP‐HPLC), and the oxidized site of glucagon was detected by electrospray ionization tandem mass spectrometry (ESI‐MS/MS). It was shown that ²⁷methionine (Met) was oxidized to ²⁷Met sulfoxide by hydroxyl radical, and the production rate of ²⁷Met sulfoxide was faster than that by hydrogen peroxide. In addition, production of ²⁷Met sulfoxide enantiomer was confirmed by RP‐HPLC analysis. cAMP production in a HepG2 cell induced by ²⁷Met sulfoxide glucagon was reduced to approximately 75% as compared with that induced by the native form of glucagon. Copyright © 2009 John Wiley & Sons, Ltd.     

An online coupled peritoneal macrophage/cell membrane chromatography and high‐performance liquid chromatography/mass spectrometry method to screen for anti‐inflammatory components from the Chinese traditional medicine Chloranthus multistachys Pei

Cell membrane chromatography (CMC) is a chromatographic biological affinity method that uses specific cell membranes as the stationary phase. In this study, a novel peritoneal macrophage/cell membrane chromatography (PM/CMC)–online‐high performance liquid chromatography/mass spectrometry (HPLC/MS) method was established to screen for the anti‐inflammatory components from traditional Chinese medicines using hydrocortisone and dexamethasone as standards. The stationary phase of the CMC employed mouse peritoneal macrophage cell membranes. This method was applied to the purification and identification of components in extracts of Chloranthus multistachys Pei. The major component retained by CMC was identified as isofraxidin by HPLC/MS. In vitro experiments revealed that IF was able to inhibit the production of nitric oxide and tumor necrosis factor‐α in lipopolysaccharide‐stimulated mice and peritoneal macrophages in a dose‐dependent manner. The results demonstrated that the PM/CMC‐online‐HPLC/MS is an effective screening system for the rapid detection, enrichment, and identification of target components from complex samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Method for rapidly discovering active components in Yupingfeng granules by UPLC‐ESI‐Q‐TOF‐MS

Yupingfeng granules (YPFG) were isolated from a traditional Chinese medicine (TCM) formulation composed of three herbs (Astragali Radix, Atractylodis Macrocephalae Rhizoma, and Saposhnikoviae Radix). This formulation is used in TCM to tonify qi, and it can help strengthen exterior and reduce sweating. Nevertheless, the active components of YPFG remain unclear. In this study, the chemical constituents of YPFG were systematically characterized by ultra‐performance liquid chromatography coupled with electrospray ionization/ quadrupole time‐of‐flight mass spectrometry (UPLC‐ESI‐Q‐TOF‐MS). Fifty‐eight compounds, namely, 20 flavonoids, 19 saponins, nine organic acids, four volatile coumarins, three lactones, one alkaloid, and two other components, were identified. In addition, the constituents of YPFG with the potential for in vivo bioactivities following oral administration were investigated in Sprague–Dawley rats. Thirteen compounds, namely, 11 flavonoid‐related and 2 saponin‐related components, were detected in rat plasma. After enriching flavonoids and saponins in YPFG by extraction, the extracts and YPFG were administrated to immunosuppressed rats, respectively. Plasma samples were analyzed by UPLC‐ESI‐Q‐TOF‐MS, and principal component analysis (PCA) confirmed that the extracts had similar effects to YPFG. This method could discover active ingredients in YPFG quickly and provide a scientific basis for quality control and mechanism research.     

Screening active anti‐breast cancer compounds from Cortex Magnolia officinalis by 2D LC–MS

Most of the anti‐breast cancer drugs are often limited owing to drug resistance and serious adverse reactions. Therefore, development of more targeted and low toxic drugs from traditional Chinese medicines for breast cancer are needed. At the same time, establishment of fast and effective drug screening methods are urgently required. We describe here a 2D LC method of MDA‐MB‐231 cell membrane chromatography combined with HPLC/MS for recognition, separation, and identification of target components from traditional Chinese medicine Cortex Magnolia officinalis. The MDA‐MB‐231 cells membrane was used to prepare the chromatographic stationary phase in the first dimension. The active compounds had a retention characteristic on the cell membrane chromatography model (10 × 2.0 mm, 5 μm). The retention fractions were enriched using an online C₁₈ column (10 × 1.0 mm, 5 μm) and were analyzed by the second dimension RP chromatography. Finally, the activity of the retention fractions was tested through in vitro experiments. Results showed that the retention fractions were honokiol and magnolol and the inhibition rate on MDA‐MB‐231 cell growth were 23 and 64 μM, respectively. These results support the conclusion that this coupled analytical technique could be an efficient method in drug discovery.     

Rapid analysis of constituents of Radix Cyathulae using hydrophilic interaction‐reverse phase LC‐MS

A hydrophilic interaction chromatography (HILIC) and reverse‐phase liquid chromatography (RPLC) coupled with electrospray TOF MS method was developed for the analysis and characterization of constituents in the radix of Cyathula officinalis Kuan. Separation parameters of HILIC such as buffer pH, mobile phase strength, and organic modifier were evaluated. Fructose, glucose, and sucrose were identified by HILIC‐ESI/TOF MS. Reverse‐phase liquid chromatography‐ESI/TOF MS were applied for quick and sensitive identification of major saponins in Cyathula officinalis. In‐source collision‐induced dissociation has been performed to elucidate the fragmentation pathways of oleanane‐, hederagenin‐, and gypsogmin‐type saponins. Twelve saponins were characterized in this plant for the first time, and four of them were presumed to be new compounds. In addition, one phytoecdysteroid (cyasterone) and one coumarin (6,7‐dimethoxycoumarin) were detected at the same time. The present method was capable of rapid characterizing and providing structure information of constituents from herbal drugs.     

Qualitative and quantitative analysis of the major constituents in Acorus tatarinowii Schott by HPLC/ESI‐QTOF‐MS/MS

Acorus tatarinowii Schott (ATS) is a well‐known traditional Chinese medicine (TCM) for the treatment of epilepsy, amnesia and insomnia. In this study, a methodology utilizing high‐performance liquid chromatography (HPLC) coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐QTOF‐MS/MS) was established for the separation and structural identification of the major chemical constituents in ATS for the first time. Overall, 46 major constituents including flavonoid glycosides, phenylpropane derivatives, amides and lignans were identified or tentatively characterized. Seven major constituents, including four phenylpropane derivatives and three lignans, were further quantified as marker substances, which showed good linearity within the test ranges. These results indicated that the developed quantitative method was linear, sensitive, and precise for quality control of ATS. Copyright © 2014 John Wiley & Sons, Ltd.     

Structures of oxaliplatin‐oligonucleotide adducts from DNA

Oxaliplatin, [(1R,2R)‐cyclohexane‐1,2‐diamine](ethanedioato‐O,O')platinum(II) shows a great efficiency against colorectal cancer. Although the mode of action of oxaliplatin is not yet understood, it is commonly accepted that binding of oxaliplatin to DNA prevents DNA synthesis and alters protein to DNA binding. In order to elucidate the modified DNA–protein interaction and thus to understand the mechanisms leading to cellular misinterpretation of DNA information and apoptosis, we have identified the preferential binding sites and the dynamics of the oxaliplatin‐DNA intrastrand and interstrand adducts at the oligomer level using high‐performance liquid chromatography/electrospray ionization‐tandem mass spectrometry (HPLC/ESI‐MS/MS) and HPLC/inductively coupled plasma‐MS for quantitative studies. We used a combination of benzonase, alkaline phosphatase and Nuclease S1 for digestion. This digestion procedure allows the study of platinated oligomeric nucleotides and more complex interstrand adducts. The digestion products were mostly chromatographically separated and characterized using HPLC/ESI‐ion trap MS/MS experiments. We could show that the adducts to guanine and adenine are quite dynamic; that is, the ratios are changing for several days. In addition, the resulting adducts provide evidence for the action of the digesting enzymes and indicate that the adduct spectrum at the oligomeric level is different to that at the commonly studies dinucleotide level. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method for simultaneous quantitative analysis of free and conjugated bisphenol A in human urine

Bisphenol A (BPA) is an environmental endocrine‐disrupting chemicals that is widely used in common consumer products. There is an increasing concern regarding human exposure to BPA owing to the potential adverse effects associated with its estrogenic activity. For assessing environmental exposure to BPA, it is essential to have a sensitive, accurate and selective analytical method, especially one that can detect low BPA levels in complex sample matrices. In this study, we developed and validated an accurate, sensitive, and robust liquid chromatography–tandem mass spectrometry method for simultaneous quantification of free BPA and BPA β‐d ‐glucuronide (BPA‐gluc) concentrations in human urine with only a single injection. Calibration curves were linear over a concentration range of 1–100 ng/mL for BPA and 10–1000 ng/mL for BPA‐gluc. The levels of the analytes were determined quantitatively with HPLC/ESI‐MS/MS by using negative electrospray ionization in the select ion monitoring mode and a pentaflouraphenyl propyl column. The validated method was applied to the analysis of spot urine specimens collected from randomly selected healthy human subjects. Copyright © 2013 John Wiley & Sons, Ltd.     

HPLC‐DAD and HPLC‐ESI‐MS separation,determination and identification of the spin‐labeled diastereoisomers of podophyllotoxin

Spin‐labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, the 2‐H configurations of these spin‐labeled derivatives cannot be determined by nuclear magnetic resonance (NMR) methods. In the present paper, a high‐performance liquid chromatography‐diode array detection (HPLC‐DAD) and a high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI/MS/MS) method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 position. In the HPLC‐ESI/MS spectra, each pair of diastereoisomers of the spin‐labeled derivatives in the mixture was directly confirmed and identified by [M+H]⁺ ions and ion ratios of relative abundance of [M‐ROH+H]⁺ (ion 397) to [M+H]⁺. When the [M‐ROH+H]⁺ ions (at m/z 397) were selected as the precursor ions to perform the MS/MS product ion scan. The product ions at m/z 313, 282, and 229 were the common diagnostic ions. The ion ratios of relative abundance of the [M‐ROH+H]⁺ (ion 397) to [M+H]⁺, [A+H]⁺ (ion 313) to [M‐ROH+H]⁺, [A+H‐OCH₃]⁺ (ion 282) to [M‐ROH+H]⁺ and [M‐ROH‐ArH+H]⁺ (ion 229) to [M‐ROH+H]⁺ of each pair of diastereoisomers of the derivatives specifically exhibited a stereochemical effect. Thus, by using identical chromatographic conditions, the combination of DAD and MS/MS data permitted the separation and identification of the four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 in the mixture.     

Analysis and Identification of the Chemical Constituents of Ding‐Zhi‐Xiao‐Wan Prescription by HPLC‐IT‐MSn and HPLC‐Q‐TOF‐MS

Ding‐Zhi‐Xiao‐Wan (DZXW) is a famous traditional Chinese medicine (TCM) formula, which is composed of four herbs, Ginseng Radix, Poria, Polygala Radix and Acori Tatarinowii Rhizoma. It has been popularly used for the treatment of emotional disease, like Alzheimer's disease, Parkinson's disease, depression, anxiety, forgetfulness and neurasthenia. In this research, a high‐performance liquid chromatography coupled with ion‐trap tandem mass spectrometry (HPLC‐IT‐MSⁿ) method along with a high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (HPLC‐Q‐TOF‐MS) method in negative ion mode was established to investigate the major constitutions in DZXW. The extracts were prepared by ultra‐sonication in ethyl acetate, n‐butanol, 95% ethanol and deionized water sequentially as well as in deionized water directly. A Kromasil C18 column was used to separate the extracts of DZXW. Acetonitrile and 0.1% aqueous formic acid (V/V) were used as the mobile phase. A total of 64 components were characterized, including 16 triterpenoids, 14 Polygala saponins, 10 oligosaccharide esters, 6 sucrose esters, 2 xanthone C‐glycosides and 16 ginsenosides.     

Quantification of intact covalently metal labeled proteins using ESI‐MS/MS

Mass spectrometric methods matured from the successful qualitative characterization of proteins in complex mixtures into methods for quantitative proteomics often based on chemical tags with stable isotope labeling. In the study presented here, we extended the application of lanthanide‐ion‐based tags from the quantification using inductively coupled plasma‐MS into the quantification of labeled intact proteins using electrospray ionization (ESI)‐MS and ESI‐MS/MS. We applied the metal chelate tag MeCAT‐iodoacetamide (IA) (1,4,7,10‐tetraazacyclododecane N,N′,N″,N″ ′‐tetra acetic acid with a IA reactive site). Labeled proteins were separated using C3‐reversed phase‐high‐performance liquid chromatography interfaced to ESI‐MS. We could prove that even large proteins were completely labeled at all available cysteine residues using MeCAT‐IA with only a small excess of reagent. Fragmentation of labeled proteins either using infrared multiphoton dissociation in Fourier transform ion cyclotron resonance‐MS or higher‐energy collision dissociation with an Orbitrap gave characteristic fragments. We used these fragments to quantify several intact proteins avoiding digestion. To demonstrate the applicability, human serum albumin was quantified in blood serum. The high‐performance liquid chromatography/ESI‐MS/MS quantification data were validated using inductively coupled plasma‐MS. Because the metal within the tag may be any of the lanthanides, multiplexing capabilities are inherent. Copyright © 2014 John Wiley & Sons, Ltd.     

A rapid HPLC‐ESI‐MS/MS method for determination of β‐asarone,a potential anti‐epileptic agent,in plasma after oral administration of Acorus calamus extract to rats

β‐Asarone (BAS), a phenylpropanoid from Acorus calamus Linn., has shown biological effects in the management of cognitive impairment conditions such as Alzheimer's disease. The present paper describes a selective and sensitive liquid chromatography–tandem mass spectrometric method (HPLC‐MS/MS) using electrospray ionization source (ESI) for quantification of BAS in rat plasma. Briefly, the plasma samples were pre‐treated using a simple solid‐phase extraction method. The separation of BAS and the internal standard, caffeine, was achieved on an Agilent Zorbax XDB C₁₈ column (50 × 2.1 mm i.d., 5 µm) using 0.2 mL/min isocratic mobile phase flow. The detection was performed using an Applied Biosystems Hybrid Q‐Trap API 2000 mass spectrometer equipped with an ESI source operated in positive mode. Also, the developed bioanalytical method was validated as per the US FDA bioanalytical guidelines over the concentration range of 9.79–4892.50 ng/mL (r² ≥ 0.9951) for BAS from rat plasma. The mean percentage recovery (n = 3) for the low, middle and high quality control samples was 86.92 ± 3.89, 85.30 ± 1.09 and 87.24 ± 4.03%, respectively. The applicability of the validated HPLC‐MS/MS method was demonstrated by successful measurement of BAS from plasma following oral administration of Acorus calamus rhizome extracts to three female albino Wistar rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Combination of virtual and experimental 2DE together with ESI LC‐MS/MS gives a clearer view about proteomes of human cells and plasma

Virtual and experimental 2DE coupled with ESI LC‐MS/MS was introduced to obtain better representation of the information about human proteome. The proteins from HEPG2 cells and human blood plasma were run by 2DE. After staining and protein spot identification by MALDI‐TOF MS, the protein maps were generated. The experimental physicochemical parameters (pI/Mw) of the proteoforms further detected by ESI LC‐MS/MS in these spots were obtained. Next, the theoretical pI and Mw of identified proteins were calculated using program Compute pI/Mw ( http://web.expasy.org/compute_pi/pi_tool‐doc.html ). Accordingly, the relationship between theoretical and experimental parameters was analyzed, and the correlation plots were built. Additionally, virtual/experimental information about different protein species/proteoforms from the same genes was extracted. As it was revealed from the plots, the major proteoforms detected in HepG2 cell line have pI/Mw parameters similar to theoretical values. In opposite, the minor protein species have mainly very different from theoretical pI and Mw parameters. A similar situation was observed in plasma in much higher degree. It means that minor protein species are heavily modified in cell and even more in plasma proteome.     

Screening and analysis of metabolites in rat urine after oral administration of Apocynum venetum L. extracts using HPLC–TOF‐MS

HPLC with diode array detection and ESI‐TOF‐MS was used for the study of the constituents in Apocynum venetum L. extracts and the metabolites in rat urine after oral administration of A. venetum L. extracts. A formula database of the known constituents in A. venetum L. was established, and 21 constituents were rapidly identified by accurately matching their molecular masses with the formulae of the compounds in the database. Furthermore, 34 metabolites were detected and elucidated in the rat urine. The scientific and plausible biotransformation pathways of the flavonoid components in A. venetum L. were also proposed together with the presentation of clues for potential mechanisms of bioactivity. This specific and sensitive HPLC–ESI‐TOF‐MS method can be used to identify the chemical components in the extracts of A. venetum L. and their metabolites in rat urine. This method can also be used to reveal the possible metabolic mechanisms of action of the extract components in vivo.