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.     

Identification and determination of the major constituents in Kai‐Xin‐San by UPLC‐Q/TOF MS and UFLC‐MS/MS method

In order to have overall chemical material information of Kai‐Xin‐San (KXS), the reliable ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometer (UHPLC–Q‐TOF‐MS) and ultra‐fast liquid chromatography mass spectrometer (UFLC‐MS/MS) methods were developed for the identification and determination of the major constituents in KXS. Moreover, the UHPLC–Q‐TOF‐MS method was also applied to screen for multiple absorbed components in rat plasma after oral administration of KXS. The UHPLC–Q‐TOF‐MS method was achieved on Agilent 6520 Q‐TOF mass and operated in the negative ion mode. Good separation was performed on a ZORBAX Eclipse Plus C18 column with a gradient elution at a flow rate of 0.2 ml/min. A total of 92 compounds in KXS were identified or tentatively characterized based on their exact molecular weights, fragmentation patterns, and literature data. A total of 26 compounds including 23 prototype components and three metabolites were identified in rat plasma after oral administration of KXS. Then, 16 major bioactive constituents were chosen as the benchmark substances to evaluate the quality of KXS. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple‐reaction monitoring mode(MRM). The analysis was completed with a gradient elution at a flow rate of 0.4 ml/min within 35 min. The simple and fast method was validated and showed good linearity, precision, and recovery. Furthermore, the method was successful applied for the determination of 16 compounds in KXS. All results would provide essential data for identification and quality control of active chemical constituents in KXS. Copyright © 2016 John Wiley & Sons, Ltd.     

Chemical UPLC‐ESI‐MS/MS profiling of aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Aconitum carmichaelii Debx. Root extract

In this paper, an ultra high performance liquid chromatography tandem mass spectrometric (UPLC‐ESI‐MS/MS) method in positive ion mode was established to systematically identify and to compare the major aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Fuzi extract. A total twenty‐nine components including twenty‐five C19‐diterpenoid alkaloids and four C20‐diterpenoid alkaloids were identified in Fuzi extract. Thirteen of the parent components and five metabolites were detected in rat plasma and sixteen parent compounds and six metabolites in urine. These parent components found in rat plasma and urine were mainly C19‐diterpenoid alkaloids. All of the metabolites in vivo were demethylated metabolites (phase I metabolites), which suggested that demethylation was the major metabolic pathway of aconitum alkaloids in vivo. A comparison of the parent components in rat plasma and urine revealed that 3‐deoxyacontine was found in plasma but not in urine, while kalacolidine, senbusine and 16‐β‐hydroxycardiopetaline existed in urine but not in plasma, which indicated that most alkaloids components were disposed and excreted in prototype form. This research provides some important information for further metabolic investigations of Fuzi in vivo.     

Identification of the absorbed components and metabolites of modified Huo Luo Xiao Ling Dan in rat plasma by UHPLC‐Q‐TOF/MS/MS

To reveal the material basis of Huo Luo Xiao Ling Dan (HLXLD), a sensitive and selective ultra‐high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF/MS) method was developed to identify the absorbed components and metabolites in rat plasma after oral administration of HLXLD. The plasma samples were pretreated by liquid–liquid extraction and separated on a Shim‐pack XR‐ODS C₁₈ column (75 × 3.0 mm, 2.2 μm) using a gradient elution program. With the optimized conditions and single sample injection of each positive or negative ion mode, a total of 109 compounds, including 78 prototype compounds and 31 metabolites, were identified or tentatively characterized. The fragmentation patterns of representative compounds were illustrated as well. The results indicated that aromatization and hydration were the main metabolic pathways of lactones and tanshinone‐related metabolites; demethylation and oxidation were the major metabolic pathways of alkaloid‐related compounds; methylation and sulfation were the main metabolic pathways of phenolic acid‐related metabolites. It is concluded the developed UHPLC‐Q‐TOF/MS method with high sensitivity and resolution is suitable for identifying and characterizing the absorbed components and metabolites of HLXLD, and the results will provide essential data for further studying the relationship between the chemical components and pharmacological activity of HLXLD.     

Analysis of the constituents in rat plasma after oral administration of Shexiang Baoxin pill by HPLC‐ESI‐MS/MS

A valid method using liquid chromatography coupled with electrospray ionization (ESI) and ion trap mass spectrometry was established for the study of the absorbed components in rat plasma after oral administration of a traditional Chinese medicine (TCM) Shexiang Baoxin pill. The plasma was deproteinated by adding methanol prior to liquid chromatography, in which separation was carried out on a Symmetry C₁₈ column (5 µm, 250 × 4.6 mm). A linear gradient with 0.5% formic acid–water–acetonitrile was used as mobile phase. Mass spectra were acquired in both negative and positive modes. Twenty‐one components including 17 components from Shexiang Baoxin pill and four metabolites were observed from a comprehensive analysis of the chromatography of Shexiang Baoxin pill, controlled plasma and dosed plasma. All of the 17 prototype compounds and three of the metabolites were identified by comparing their retention behaviors and MS and MS/MS spectra with reference compounds and literature data. This study developed an integrated method for screening the bioactive constituents in plasma after oral adminstration of Chinese herbal medicine and provided helpful chemical information for further pharmacology and active mechanism research on TCM. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of chemical constituents in Rhizoma Paridis Saponins and their oral administration in rat plasma by UPLC/Q‐TOF/MS

On‐line ultra‐performance liquid chromatography (UPLC) coupled with diode‐array detection (UPLC/DAD) and electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF‐MS) were used for separation, identification and structural analyses of saponins in Rhizoma Paridis saponins (RPS) and rat plasma after oral administration of RPS. Thirty steroidal saponins in RPS were identified by comparing their retention time, accurate mass measurement and positive and negative mass spectrometry data with that of reference compounds. The UPLC/Q‐TOF‐MS method was proved to be rapid and efficient in that 30 steroidal saponins, including three kinds of saponins (prototype, pennogenyl and diosgenyl saponins) were tentatively characterized within 6 min. After oral administration of RPS, 21 original saponins were absorbed in RPS‐treated rat plasma. Our results indicated that UPLC/Q‐TOF‐MS is a rapid and effective tool for identification of a series of saponins at trace level. Copyright © 2010 John Wiley & Sons, Ltd.     

UPLC–Q‐TOF–MS and UPLC–MS/MS methods for metabolism profiles and pharmacokinetics of major compounds in Xuanmai Ganjie Granules

Xuanmai Ganjie Granules (XMGJ), a widely used Chinese herbal formula in the clinic, is used for treatment of sore throats and coughs. Despite the chemical constituents having been clarifying by our previous studies, both of the metabolism and pharmacokinetic studies of XMGJ are unclear. This study aimed to explore the disposition process of XMGJ in vivo. A sensitive and selective ultra‐high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry (UPLC–Q‐TOF–MS) method was developed to analyze the absorbed components and metabolites in rat plasma and urine after oral administration of XMGJ. A total of 42 absorbed components, including 16 prototype compounds and 26 metabolites, were identified or tentatively characterized in rat plasma and urine after oral administration of XMGJ. Moreover, the pharmacokinetic studies of five compounds of XMGJ were investigated using ultra‐high liquid chromatography with tandem mass spectrometry method. The results indicated that liquiritin, harpagoside, glycyrrhetic acid, liquiritigenin, formononetin and their metabolites might be the major components involved in the pharmacokinetic and metabolism process of XMGJ. This research showed a comprehensive investigation of XMGJ in vivo, which could provide a meaningful basis for further material basis and pharmacological as well as toxicological research.     

New cathinone‐derived designer drugs 3‐bromomethcathinone and 3‐fluoromethcathinone: studies on their metabolism in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS and their detectability in urine

3‐Bromomethcathinone (3‐BMC) and 3‐Fluoromethcathinone (3‐FMC) are two new designer drugs, which were seized in Israel during 2009 and had also appeared on the illicit drug market in Germany. These two compounds were sold via the Internet as so‐called “bath salts” or “plant feeders.” The aim of the present study was to identify for the first time the 3‐BMC and 3‐FMC Phase I and II metabolites in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS (HR‐MS) and to test for their detectability by established urine screening approaches using GC–MS or LC–MS. Furthermore, the human cytochrome‐P450 (CYP) isoenzymes responsible for the main metabolic steps were studied to highlight possible risks of consumption due to drug–drug interaction or genetic variations. For the first aim, rat urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified by GC–MS and by LC–HR‐MS. The main metabolic steps were N‐demethylation, reduction of the keto group to the corresponding alcohol, hydroxylation of the aromatic system and combinations of these steps. The elemental composition of the metabolites identified by GC–MS could be confirmed by LC–HR‐MS. Furthermore, corresponding Phase II metabolites were identified using the LC–HR‐MS approach. For both compounds, detection in rat urine was possible within the authors' systematic toxicological analysis using both GC–MS and LC–MSⁿ after a suspected recreational users dose. Following CYP enzyme kinetic studies, CYP2B6 was the most relevant enzyme for both the N‐demethylation of 3‐BMC and 3‐FMC after in vitro–in vivo extrapolation. Copyright © 2012 John Wiley & Sons, Ltd.     

Studies on the metabolism of the Δ9‐tetrahydrocannabinol precursor Δ9‐tetrahydrocannabinolic acid A (Δ9‐THCA‐A) in rat using LC‐MS/MS,LC‐QTOF MS and GC‐MS techniques

In Cannabis sativa, Δ9‐Tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A) is the non‐psychoactive precursor of Δ9‐tetrahydrocannabinol (Δ9‐THC). In fresh plant material, about 90% of the total Δ9‐THC is available as Δ9‐THCA‐A. When heated (smoked or baked), Δ9‐THCA‐A is only partially converted to Δ9‐THC and therefore, Δ9‐THCA‐A can be detected in serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of Δ9‐THCA‐A and to examine particularly whether oral intake of Δ9‐THCA‐A leads to in vivo formation of Δ9‐THC in a rat model. After oral application of pure Δ9‐THCA‐A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identified by liquid chromatography‐mass spectrometry (LC‐MS), liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and high resolution LC‐MS using time of flight‐mass spectrometry (TOF‐MS) for accurate mass measurement. For detection of Δ9‐THC and its metabolites, urine extracts were analyzed by gas chromatography‐mass spectrometry (GC‐MS). The identified metabolites show that Δ9‐THCA‐A undergoes a hydroxylation in position 11 to 11‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A (11‐OH‐Δ9‐THCA‐A), which is further oxidized via the intermediate aldehyde 11‐oxo‐Δ9‐THCA‐A to 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A‐COOH). Glucuronides of the parent compound and both main metabolites were identified in the rat urine as well. Furthermore, Δ9‐THCA‐A undergoes hydroxylation in position 8 to 8‐alpha‐ and 8‐beta‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A, respectively, (8α‐Hydroxy‐Δ9‐THCA‐A and 8β‐Hydroxy‐Δ9‐THCA‐A, respectively) followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Several glucuronidation conjugates of these metabolites were identified. In vivo conversion of Δ9‐THCA‐A to Δ9‐THC was not observed. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of the constituents and metabolites in rats after oral administration of Zi Shen Formula by UPLC‐Q‐TOF/MS combined pattern recognition analysis

An ultra‐high‐performance liquid chromatography mass spectrometry method was established to detect and identify the chemical constituents of Zi Shen Formula (ZSF) and its metabolites in serum, urine and feces, after oral administration to rats. A total of 68 compounds were characterized in ZSF extracts. In vivo, 38 prototype components and 32 metabolites of ZSF were tentatively identified in rat serum, urine and feces. Seven metabolic pathways including demethylation, hydroxylation, oxidation, sulfation, glucuronidation, methylation and de‐caffeoyl were proposed to be involved in the generation of these metabolites. It was found that glucuronidation, methylation and demethylation were the major metabolic processes of alkaloids, while demethylation, methylation, sulfation and de‐caffeoyl were the major metabolic pathways of phenylethanoid glycosides. The main metabolic pathways of steroidal saponins were oxidation and isotype reactions. These findings are significant for our understanding of the metabolism of ZSF. The proposed metabolic pathways of bioactive components might be crucial for further studies of the mechanisms of action and pharmacokinetic evaluations of ZSF.     

Analysis of the metabolic profile of parishin by ultra‐performance liquid chromatography/quadrupole‐time of flight mass spectrometry

Parishin is a dominant active ingredient originating from Gastrodia elata Blume, and has good neuroprotective effects against brain disorders. In the present study, the metabolic profile of parishin by in vitro and in vivo experiments was investigated using ultra‐high performance liquid chromatography coupled with quadrupole–time of flight mass spectrometry (UHPLC/Q‐TOF MS) combined with an automated MS^E technique. By comparison with reference compounds, accurate mass measurement, the characteristic fragmentation patterns of the parent drug parishin and gastrodin and relevant bio‐transformation knowledge, 14 metabolites (seven hydrolyzates and seven derivatives of gastrodin) were detected and identified in rat plasma and urine after intragastric administration of parishin, including processes of hydrolyzation, oxidation, sulfation and glucuronidation. According to the proposed metabolic pathways of parishin, in vitro hydrolytic experiments and metabolic study of gastrodin in rat plasma, it can be inferred that parishin mainly functions as a prodrug and undergoes hydrolysis before being absorbed into the blood. The hydrolyzate, mainly gastrodin, was involved in further metabolism, which was responsible for pharmacological activities of parishin. In conclusion, this work provides valuable information on parishin metabolism using a rapid and reliable UHPLC/Q‐TOF MS method, which could be widely used for the metabolic investigation of natural product. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of the major urinary metabolites in man of seven synthetic cannabinoids of the aminoalkylindole type present as adulterants in ‘herbal mixtures’ using LC‐MS/MS techniques

Herbal mixtures, such as ‘Spice’, containing cannabimimetic compounds are easily available on the Internet and have become increasingly popular among people having to undergo urine drug testing, as these compounds are not detected by current immunochemical tests. For analysis of urine samples, knowledge of the main metabolites is necessary as the unchanged compounds are usually not found in urine after consumption. In this paper, the identification of the major metabolites of the currently most common seven synthetic cannabinoids is presented. Urine samples from patients of psychiatric facilities known to have consumed synthetic cannabinoids were screened by LC‐MS/MS and HR‐MS/MS techniques, and the major metabolites for each of the following synthetic cannabinoids were identified by their enhanced product ion spectra and accurate mass measurement: JWH‐018, JWH‐073, JWH‐081, JWH‐122, JWH‐210, JWH‐250 and RCS‐4. The major metabolic pathway is monohydroxylation either at the N‐alkyl side chain, the naphthyl moiety or the indole moiety. In addition, metabolites with carboxylated alkyl chains were identified for some of the compounds. These results facilitate the design of urine screening methods for detecting consumption of synthetic cannabinoids. Copyright © 2012 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.     

Chemical fingerprint analysis and metabolic profiling of 50% ethanol fraction of Lomatogonium rotatum by ultra‐performance liquid chromatography/quadrupole–time of flight mass spectrometry

Lomatogonium rotatum (L.) Fries ex Nym (L. rotatum), a member of Gentianaceae, is an important mongolian medicine in China used to treat febrile diseases in liver and gallbladder. The aim of present study was to investigate the chemical constituents and metabolites of the 50% ethanol fraction of L. rotatum (50EtLR). Firstly, the extract of L. rotatum was partitioned by macroporous resin to obtain the target fraction (50EtLR), then several compounds were isolated from 50EtLR to obtained the standards for further analysis of chemical constituents of 50EtLR. Secondly, the chemical constituents of 50EtLR were characterized using the ultra‐high performance liquid chromatography coupled with quadrupole–time‐of‐flight mass spectrometry (UHPLC–Q‐TOF–MS/MS). Finally, prototype constituents and related metabolites were analyzed after orally administerng 50EtLR to rats. As a result, a new compound, 6‐O‐[β‐d ‐xylopyranosyl‐(1 → 6)‐O‐β‐d ‐glucopyranosyl]‐1,4,8‐trimethoxyxanthone ( 6 ) along with seven known compounds ( 1–5 , 7 and 8 ) were isolated from the 50EtLR, 92 components were either unambiguously or tentatively identified. Additionally, 34 prototype constituents and 112 metabolites in rat plasma along with 32 prototype constituents and 53 metabolites in rat liver were tentatively identified. Therefore, xanthones and flavonoids were the main chemical constituents of 50EtLR and sulfation and glucuronidation are the main enzyme‐induced metabolic pathways involved post‐administration.     

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.     

Metabolite analysis in Curcuma domestica using various GC‐MS and LC‐MS separation and detection techniques

The metabolic profile of polar (methanol) and non‐polar (hexane) extracts of Curcuma domestica, a widely used medicinal plant, was established using various different analytical techniques, including GC‐FID, GC‐MS, HR‐GC‐MS and analytical HPLC‐ESI‐MS/MS by means of LTQ‐Orbitrap technology. The major non‐volatile curcuminoids curcumin, demethoxycurcumin and bisdemethoxycurcumin were identified when their chromatographic and precursor ion masses were compared with those of authentic standard compounds. In this paper we describe for the first time a GC/MS‐based method for metabolic profiling of the hydrophilic extract. We also identified 61 polar metabolites as TMS derivatives. Copyright © 2009 John Wiley & Sons, Ltd.     

GC‐MS of amaryllidaceous galanthamine‐type alkaloids

Galanthamine‐type alkaloids produced by plants of the Amaryllidaceae family are potent acetylcholinesterase inhibitors. One of them, galanthamine, has been marketed as a hydrobromide salt for the treatment of Alzheimer's disease. In the present work, gas chromatography with electron impact mass spectrometry (GC‐EIMS) fragmentation of 12 reference compounds isolated from various amaryllidaceous plants and identified by spectroscopic methods (1D and 2D nuclear magnetic resonance, circular dichroism, high‐resolution MS (HRMS) and EIMS) was studied by tandem mass spectrometry (GC‐MS/MS) and accurate mass measurements (GC‐HRMS). The studied compounds showed good peak shape and efficient GC separation with a GC‐MS fragmentation pattern similar to that obtained by direct insertion probe. With the exception of galanthamine‐N‐oxide and N‐formylnorgalanthamine, the galanthamine‐type compounds showed abundant [M]^+. and [M‐H]⁺ ions. A typical fragmentation pattern was also observed, depending on the substituents of the skeleton. Based on the fragmentation pathways of reference compounds, three other galanthamine‐type alkaloids, including 3‐O‐(2′‐butenoyl)sanguinine, which possesses a previously unelucidated structure, were identified in Leucojum aestivum ssp. pulchelum, a species endemic to the Balearic islands. GC‐MS can be successfully applied to Amaryllidaceae plant samples in the routine screening for potentially new or known bioactive molecules, chemotaxonomy, biodiversity and identification of impurities in pharmaceutical substances. Copyright © 2012 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.