Identification of metabolites of Helicid in vivo using ultra‐high performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry

Helicid is an active natural aromatic phenolic glycoside ingredient originating from a well‐known traditional Chinese herbal medicine and has the significant effects of sedative hypnosis, anti‐inflammatory analgesia and antidepressant. In this study, we analyzed the potential metabolites of Helicid in rats by multiple mass defect filter and dynamic background subtraction in ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF‐MS). Moreover, we used a novel data processing method, ‘key product ions’, to rapidly detect and identify metabolites as an assistant tool. MetabolitePilot™ 2.0 software and PeakView™ 2.2 software were used for analyzing metabolites. Twenty metabolites of Helicid (including 15 phase I metabolites and five phase II metabolites) were detected by comparison with the blank samples. The biotransformation route of Helicid was identified as demethylation, oxidation, dehydroxylation, hydrogenation, decarbonylation, glucuronide conjugation and methylation. This is the first study simultaneously detecting and identifying Helicid metabolism in rats employing UHPLC‐Q‐TOF‐MS technology. This experiment not only proposed a method for rapidly detecting and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of Helicid in vivo. Furthermore, it provided an effective method for the analysis of other aromatic phenolic glycosides metabolic components in vivo.     

Multi‐analysis strategy for metabolism of Andrographis paniculata in rat using liquid chromatography/quadrupole time‐of‐flight mass spectrometry

Compared with chemical drugs, it is a huge challenge to identify active ingredients of multicomponent traditional Chinese medicine (TCM). For most TCMs, metabolism investigation of absorbed constituents is a feasible way to clarify the active material basis. Although Andrographis paniculata (AP) has been extensively researched by domestic and foreign scholars, its metabolism has seldom been fully addressed to date. In this paper, high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry was applied to analysis and characterization of AP metabolism in rat urine and feces samples after oral administration of ethanol extract. The differences in metabolites and metabolic pathways between the two biological samples were further compared. The chemical structures of 20 components were tentatively identified from drug‐treated biological samples, including six prototype components and 14 metabolites, which underwent such main metabolic pathways as hydrolyzation, hydrogenation, dehydroxylation, deoxygenation, methylation, glucuronidation, sulfonation and sulfation. Two co‐existing components were found in urine and feces samples, suggesting that some ingredients' metabolic processes were not unique. This study provides a comprehensive report on the metabolism of AP in rats, which will be helpful for understanding its mechanism. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic profiles of dioscin in rats revealed by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Dioscin (DIS), one of the most abundant bioactive steroidal saponins in Dioscorea sp., is used as a complementary medicine to treat coronary disease and angina pectoris in China. Although the pharmacological activities and pharmacokinetics of DIS have been well demonstrated, information regarding the final metabolic fates is very limited. This study investigated the in vivo metabolic profiles of DIS after oral administration by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry method. The structures of the metabolites were identified and tentatively characterized by means of comparing the molecular mass, retention time and fragmentation pattern of the analytes with those of the parent compound. A total of eight metabolites, including seven phase I and one phase II metabolites, were detected and tentatively identified for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. In addition, a possible metabolic pathway on the biotransformation of DIS in vivo was proposed. This study provides valuable and new information on the metabolism of DIS, which will be helpful for further understanding its mechanism of action. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of chemical constituents and rats metabolites of Shuanghua Baihe tablets by HPLC‐Q‐TOF‐MS/MS

A high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry method was established to detect as many constituents in rat biological fluids as possible after oral administration of Shuanghua Baihe tablets (SBT). An Agilent Poroshell 120 EC‐C₁₈ column was adopted to separate the samples, and mass spectra were acquired in positive and negative modes. First, the fingerprints of SBT were established, resulting in 32 components being detected within 40 min. Among these compounds, 12 were tentatively identified by comparing the retention times and mass spectral data with those of reference standards and the reference literature; the other 20 components were tentatively assigned solely based on the MS data. Furthermore, metabolites in rat plasma and urine after oral administration of SBT were also analyzed. A total of 19 compounds were identified, including 13 prototypes and six metabolites through metabolic pathways of demethylation and glucuronide conjugation. Glucuronidated alkaloids were the main constituents in the plasma, and were then excreted from urine. This is the first systematic study on the metabolic profiling of SBT. Copyright © 2014 John Wiley & Sons, Ltd.     

The in vivo absorbed constituents and metabolites of Danshen decoction in rats identified by HPLC with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry

Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge, is widely used for the treatment of cardiovascular and cerebrovascular diseases. This research focuses on the in vivo metabolism of Danshen decoction (DSD) in rats. After oral administration of DSD, the absorptive constituents and their metabolites in urine and plasma were analyzed by HPLC coupled with a photodiode array detector and electrospray ionization hybrid ion trap and time‐of‐flight mass spectrometry. Samples were separated on a C₁₈ column by gradient elution using 0.1% (v/v) aqueous formic acid and acetonitrile. As a result, 93 compounds from urine and 38 compounds from plasma were identified. Among them, lipo‐soluble diterpenoids (24 in urine and 15 in plasma) were reported for the first time as in vivo metabolites of DSD. According to the quantities and contents of the identified compounds, tanshinone IIA, cryptotanshinone and tanshinone I were deduced to be the major absorptive diterpenoids of DSD. Moreover, nine water‐soluble phenolics (caffeic acid, ferulic acid, danshensu, etc.) were proved to be the major absorptive constituents as reported. Most of the absorbed constituents underwent sulfation, glucuronidation, hydrogenation and hydroxylation in vivo. This investigation provided scientific evidence to obtain a more comprehensive metabolic profile of DSD. Copyright © 2014 John Wiley & Sons, Ltd.     

In vivo metabolite profiles of isoimperatorin and phellopterin in rats analyzed using HPLC coupled with diode array detector and electrospray ionization ion trap time‐of‐flight mass spectrometry technique

Isoimperatorin (IP) and phellopterin (PP) are two furocoumarins existing in Angelicae Dahuricae Radix. There is an isopentenyloxyl substituted at C‐5 in IP, and an isopentenyloxyl and a methoxyl substituted at C‐8 and C‐5, respectively, in PP. To elucidate the in vivo metabolic characteristics of PP and IP, HPLC coupled with diode array detector and electrospray ionization ion trap time‐of‐flight mass spectrometry technique was used. In total, 111 metabolites, including 53 new ones, were identified from the urine and plasma samples of rats after oral administration of IP and PP, respectively. The metabolites were formed through eight reactions on IP and PP: oxidation, hydroxylation–hydrogenation, carboxylation on the isopentenyloxyl, O‐dealkylation, hydroxylation on the furocoumarin nucleus, ring‐opening reaction on the furan ring and reduction or ring‐opening reaction on the lactone ring. Among these, hydroxylation on the furocoumarin nucleus was found for the first time for in vivo metabolites of PP and IP, and the ring‐opening reaction on the furan ring or lactone ring was found for the first time for in vivo metabolites of isopentenyloxyl furocoumarins. The research gave us a new insight into the in vivo metabolic profiles of IP and PP, which could help us better understand their important roles as two active constituents of Angelicae Dahuricae Radix.     

Identification of chemical constituents and rat metabolites of Kangxianling granule by HPLC‐Q‐TOF‐MS/MS

A high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass tandem mass spectrometry method was established to characterize the chemical constituents of Kangxianling granule (KXL), a traditional Chinese medicine formula, and the metabolic profile in rat urine and plasma after oral administration of KXL. A total of 27 compounds in KXL extract and 13 prototype compounds with 12 metabolites in rat urine and plasma were identified. Among the 27 detected compounds, 15 were identified by comparing the retention time and MS data with that of reference compounds and the other 12 compounds were tentatively assigned based on the MS data and reference literature. The main prototype components absorbed in rat were amygdalin, salvianolic acid B, tanshinones and anthraquinones. Hydroxylation, glucuronidation and sulfation were the principal metabolic pathways in rat. The results revealed that the 25 compounds identified in rat urine and plasma were the potential active ingredients of KXL, which provides helpful chemical information for further study of the pharmacology mechanism of KXL. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of absorbed constituents and in vivo metabolites in rats after oral administration of Physalis alkekengi var. franchetii by ultra‐high‐pressure liquid chromatography quadrupole time‐of‐flight mass spectrometry

The calyces of Physalis alkekengi var. franchetii (Chinese Lantern, JDL) are well‐known as traditional Chinese medicine owing to its various therapeutic effects. However, the bioactive constituents responsible for the pharmacological effects of JDL and their metabolites in vivo are still unclear to date. In this paper, an ultra‐high‐pressure liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (UHPLC/Q‐TOF‐MS/MS) method was established to identify absorbed constituents and in vivo metabolites in rat biological fluids after oral administration of JDL. Based on the proposed strategy, 33 compounds were observed in dosed rat biosamples. Twelve of 33 compounds were indicated as prototype components of JDL, and 21 compounds were predicted to be metabolites of JDL. Finally, the metabolic pathways were proposed, which were glucuronidation, sulfation, methylation and dehydroxylation for flavonoid constituents and sulfonation and hydroxylation for physalin consitituents. This is the first systematic study on the absorbed constituents and metabolic profiling of JDL and will provide a useful template for screening and characterizing the ingredients and metabolites of traditional Chinese medicine.     

Fragmentation patterns study of iridoid glycosides in Fructus Gardeniae by HPLC‐Q/TOF‐MS/MS

Iridoid glycosides (IGs), the major constituents in Fructus Gardeniae, have demonstrated various pharmacological activities, but there is no systematic chemical profile of IGs in Fructus Gardeniae in the published literature until now. Therefore, it is imperative that a rapid and sensitive high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐Q/TOF‐MS/MS) method is established for comprehensive characterization of IGs in Fructus Gardeniae. Firstly, the fragmentation patterns of six known IGs were investigated and proposed and further concluded the diagnostic fragment ions and characteristic fragmentation pathways. Then, based on the summarized fragmentation patterns and the known compounds in the literatures, the other IGs in Fructus Gardeniae were identified successively. As a result, a total of 20 IGs were identified, of which three pairs of epimers were structurally characterized and differentiated. More importantly, one compound, the isoshanzhiside methyl ester, was tentatively identified as a new compound. The results of this study demonstrate the superiority of HPLC‐MS with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of the multiple groups of constituents in Fructus Gardeniae. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolites identification of Huo Luo Xiao Ling Dan in rat urine by UPLC coupled with electrospray ionization time‐of‐flight mass spectrometry

Huo Luo Xiao Ling Dan (HLXLD), a Chinese herbal formula, is used in folk medicine for the treatment of arthritis and other chronic inflammatory diseases. However, the in vivo integrated metabolism of its multiple components remains unknown. In this paper, an ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐Q‐TOF‐MS) method was developed for detection and identification of HLXLD metabolites in rat urine at high and normal clinical dosages. The prototype constituents and their metabolites in urine were analyzed. The mass measurements were accurate within 8 ppm, and subsequent fragment ions offered higher quality structural information for interpretation of the fragmentation pathways of various compounds. A total of 85 compounds were detected in high dosages urine samples by a highly sensitive extracted ion chromatograms method, including 31 parent compounds and 54 metabolites. Our results indicated that phase 2 reactions (e.g. glucuronidation, glutathionidation and sulfation) were the main metabolic pathways of lactones, alkaloids and flavones, while phase I reactions (e.g. hydrogenation and hydroxylation) were the major metabolic reaction for coumarins, paeoniflorin and iridoids. This investigation provided important structural information on the metabolism of HLXLD and provided scientific evidence to obtain a more comprehensive metabolic profile. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of the constituents in rat plasma after oral administration of radix polygoni multiflori extracts by ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry

An ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry method was established to detect as many constituents in rat plasma as possible after oral administration of Radix polygoni multiflori (RPM) extract. A C₁₈ column (150 × 2.0 mm, 4 µm) was adopted to separate the samples, and mass spectra were acquired in negative modes. The fingerprints of RPM extract were established, resulting in 39 components being detected. Among these compounds, 29 were identified by comparing the retention times and mass spectral data with those of reference standards and relevant references, and eight compounds were separated and detected in RPM for the first time. In vivo, 23 compounds were observed in dosed rat plasma, 16 of 23 compounds were indicated as prototype components of RPM, and seven compounds were predicted to be metabolites of RPM. A high‐speed and sensitive method was developed and was successfully utilized for screening and characterizing the ingredients and metabolites of RPM. Copyright © 2015 John Wiley & Sons, Ltd.     

In vivo metabolite identification of acotiamide in rats using ultra‐performance liquid chromatography–quadrupole/time‐of‐flight mass spectrometry

Acotiamide hydrochloride (ACT) is a drug used for the treatment of functional dyspepsia. Understanding which metabolites are likely to be formed in vivo is essential for interpreting pharmacology, pharmacokinetic and toxicology data. The metabolism of ACT has been investigated using a specific and sensitive liquid chromatography positive ion electrospray ionization high‐resolution tandem mass spectrometry method. In vivo samples including rat plasma, urine and feces were collected separately after dosing healthy Sprague–Dawley rats at a dose of 20 mg kg ⁻¹ ACT at different time points up to 24 h. The metabolites were enriched by optimized sample preparation involving protein precipitation using acetonitrile followed by solid‐phase extraction. The mass defect filter technique was used for better detection of both predicted and unexpected drug metabolites with the majority of interference ions removed. The structural elucidation of the metabolites was performed by comparing their [M + H]⁺ ions and their product ions with those of the parent drug. As a result, a total of seven hitherto unknown metabolites were characterized from the biosamples. The only phase I metabolite detected was N‐ despropyl acotiamide, whereas six phase II glucuronide conjugate metabolites were identified.     

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.     

Metabolic study of paeoniflorin and total paeony glucosides from Paeoniae Radix Rubra in rats by high‐performance liquid chromatography coupled with sequential mass spectrometry

A clear understanding of the metabolism of Traditional Chinese Medicines is extremely important in their rational clinical application and effective material foundation research. A novel and reliable strategy was performed to find more metabolites of paeoniflorin, determine the metabolites of total paeony glucosides (TPG) by means of determining those metabolites of paeoniflorin, and compare the metabolism differences between paeoniflorin and TPG by intragastric administration. This strategy was characterized as follows. Firstly, the rats were divided into two groups (the paeoniflorin group and the TPG group) to find differences in metabolism mechanisms between paeoniflorin and TPG. Secondly, UPLC‐FT‐ICR MS and UPLC‐Q‐TOF MS² were applied to obtain accurate molecular weight and structural information, respectively. Thirdly, the metabolites were tentatively identified by a combination of data‐processing methods including mass defect screening, characteristic neutral loss screening and product ion screening. Finally, a comparative study was employed in the metabolism of paeoniflorin and TPG. Based on the strategy, 18 metabolites of paeoniflorin (including four new compounds) and 11 metabolites of TPG (including two new compounds) were identified. In all of the identified metabolites of paeoniflorin, two metabolites in rat plasma, four metabolites in rat urine and six metabolites in rat feces were found for the first time after paeoniflorin administration. The results indicate that hydrolyzation of the ester bond and glucosidic band and conjugation with glucuronide were the major metabolic pathways of paeoniflorin. The metabolites of paeoniflorin and TPG in rat plasma, urine and feces have been detected for the first time after intragastric administration. The results may contribute to a better understanding of the metabolism mechanism and provide a scientific rationale for researching the material basis of paeoniflorin and TPG in vivo.     

Metabolite identification of tanshinol borneol ester in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight mass spectrometry

Tanshinol borneol ester (DBZ) is a potential drug candidate composed of danshensu and borneol. It shows anti‐ischemic and anti‐atherosclerosis activity. However, little is known about its metabolism in vivo. This research aimed to elucidate the metabolic profile of DBZ through analyzing its metabolites using high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight mass spectrometry. Chromatographic separation was performed on an Agilent TC‐C₁₈ column (150 × 4.6 mm, 5.0 μm) with gradient elution using methanol and water containing 0.2% (v/v) formic acid as the mobile phase. Metabolite identification involved analyzing the retention behaviors, changes in molecular weights and MS/MS fragment patterns of DBZ and its metabolites. As a result, 20 potential metabolites were detected and tentatively identified in rat plasma, urine and feces after administration of DBZ. DBZ could be metabolized to O‐methylated DBZ, DBZ‐O‐glucuronide, O‐methylated DBZ‐O‐glucuronide, hydroxylated DBZ and danshensu. Danshensu, a hydrolysis product of DBZ, could further be transformed into 12 metabolites. The proposed method was confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of DBZ and providing valuable information on its druggability.     

Screening free radical scavengers in Xiexin Tang by HPLC‐ABTS‐DAD‐Q‐TOF/MS

Xiexin Tang (XXT) is a traditional Chinese medicine (TCM) that has been used in herbal clinics for more than 1800 years. Many studies have shown that XXT has therapeutic effects on patients with arteriosclerosis owing to its antioxidant activity. However, there is little information about the relationship between the chemical composition of XXT and its antioxidant activity. In this study, the HPLC‐ABTS‐DAD‐Q‐TOF/MS method, which can simultaneously identify individual components and rapidly screen for antioxidant compounds, was used to screen and identify antioxidant components in XXT. The 15 compounds identified were gluco‐syringic acid, adenine, gallic acid, biflorin, cularine, 6‐C ‐arabinose‐8‐C ‐glucose‐chrysin, 6‐C ‐glucose‐8‐C ‐arabinose–chrysin, baicalin, rhein‐8‐O‐β ‐d ‐glucopyranoside, coptisine, epiberberine, jatrorrhizine, norwogonin, 5,7,2′‐trihydroxy‐6‐ methoxyflavone and baicalein. In addition, the data showed that the antioxidant activity of peaks 4, 6, and 11 was lower in XXT than in its constituent herbs, while the activity of peaks 1, 2, 3, 5, 7, 8, 10, 12, 13, 14 and 15 was higher in XXT. Compound 5 had the strongest antioxidant activity in XXT, while compound 1 showed the strongest antioxidant activity among its constituent herb. The differences between antioxidant activities of major components of XXT and those of its constituent herbs might be due to the interaction of crude drugs that changes the solubility of active components during the decoction process. The results show that the HPLC‐ABTS‐DAD‐Q‐TOF/MS method can successfully combine on‐line mass spectrometry with activity detection system. It is a useful tool for the rapid detection and identification of antioxidants, and for quantitative analysis of individual antioxidants in complex mixtures such as plant extracts. Furthermore, this method does not require extensive extract purification and fraction collection.     

Pharmacokinetics and excretion study of bergenin and its phase II metabolite in rats by liquid chromatography tandem mass spectrometry

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for the determination of bergenin and its phase II metabolite in rat plasma, bile and urine has been developed. Biological samples were pretreated with protein precipitation extraction procedure and enzymatic hydrolysis method was used for converting glucuronide metabolite to its free form bergenin. Detection and quantitation were performed by MS/MS using electrospray ionization and multiple reaction monitoring. Negative electrospray ionization was employed as the ionization source. Sulfamethoxazole was used as the internal standard. The separation was performed on a reverse‐phase C₁₈ (250 × 4.6 mm, 5 μm) column with gradient elution consisting of methanol and 0.5% aqueous formic acid. The concentrations of bergenin in all biological samples were in accordance with the requirements of validation of the method. After oral administration of 12 mg/kg of the prototype drug, bergenin and its glucuronide metabolite were determined in plasma, bile and urine. Bergenin in bile was completely excreted in 24 h, and the main excreted amount of bergenin was 97.67% in the first 12 h. The drug recovery in bile within 24 h was 8.97%. In urine, the main excreted amount of bergenin was 95.69% in the first 24 h, and the drug recovery within 24 h was <22.34%. Total recovery of bergenin and its glucuronide metabolite was about 52.51% (20.31% in bile within 24 h, 32.20% in urine within 48 h). The validated method was successfully applied to pharmacokinetic and excretion studies of bergenin.     

Characterization of isochlorogenic acid A metabolites in rats using high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry

Isochlorogenic acid A is widely present in fruits, vegetables and herbal medicines, and is characterized by anti‐inflammatory, hepatoprotective and antiviral properties. However, little is known about its metabolic fate and pharmacokinetic properties. This study is thus designed to investigate the metabolic fate of isochlorogenic acid A. An analytical method based on high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (HPLC/Q‐TOF MS) was established to characterize the metabolites of isochlorogenic acid A in the plasma, urine and feces of rats. A total of 32 metabolites were identified. The metabolic pathways mainly include hydrolyzation, dehydroxylation, hydrogenation and conjugation with methyl, glucuronic acid, glycine, sulfate, glutathione and cysteine. Moreover, the pharmacokinetic profiles of all the circulating metabolites were investigated. M11 resulting from hydrolyzation, dehydroxylation and hydrogenation was the dominant circulating metabolite after the intragastric administration of isochlorogenic acid A. The results obtained will be useful for further study of elucidating potential bioactive metabolites which can provide better explanation of the pharmacological and/or toxicological effects of this compound.     

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.