Biotransformation and metabolic profile of caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐cymaropyranoside with human intestinal microflora by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In our previous studies, caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐ cymaropyranoside (CDMC) was for the first time isolated from Cynanchum auriculatum Royle ex Wightand and was reported to possess a wide range of biological activities. However, the routes and metabolites of CDMC produced by intestinal bacteria are not well understood. In this study, ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) technique combined with Metabolynx^TMsoftware was applied to analyze metabolites of CDMC by human intestinal bacteria. The incubated samples collected for 48 h in an anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF‐MS within 12 min. Eight metabolites were identified based on MS and MS/MS data. The results indicated that hydrolysis, hydrogenation, demethylation and hydroxylation were the major metabolic pathways of CDMC in vitro. Seven strains of bacteria including Bacillus sp. 46, Enterococcus sp. 30 and sp. 45, Escherichia sp. 49A, sp. 64, sp. 68 and sp. 75 were further identified using 16S rRNA gene sequencing owing to their relatively strong metabolic capacity toward CDMC. The present study provides important information about metabolic routes of CDMC and the roles of different intestinal bacteria in the metabolism of CDMC. Moreover, those metabolites might influence the biological effect of CDMC in vivo, which affects the clinical effects of this medicinal plant. Copyright © 2015 John Wiley & Sons, Ltd.     

Profiling and identification of the absorbed constituents and metabolites of schisandra lignans by ultra‐performance liquid chromatography coupled to mass spectrometry

Schisandra chinensis Baill grows wild in Russia, China, Korea and Japan, and its fruit has been found to be effective in amnesia and insomnia. It is enriched in schisandra lignans (SL) that are major components responsible for therapeutic action. However, there are no reports on the biotransformation analysis of SL. An ultra‐performance liquid chromatography/electrospray‐ionization high‐definition mass spectrometry (UPLC‐Q‐TOF‐HDMS) method was developed to investigate the metabolism of SL in vivo. MS was performed on a Waters Micromass high‐definition system with an electrospray ionization source in positive ion mode and automated MetaboLynx software analysis with excellent MS accuracy and enhanced MS data acquisition. An improved mass defect filter (MDF) method employing both drug and core structure filter templates was applied to the processing of UPLC‐Q‐TOF‐HDMS data for the detection and structural characterization of metabolites. In this study, 30 metabolites were detected and identified in vivo, and demethylation and hydroxylation were confirmed as the primacy metabolic pathway for SL in rat plasma. In conclusion, the presently developed methodology was suitable for biotransformation research of SL and will find wide use in metabolic studies for other herbal medicines. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of metabolites in rats after intravenous administration of salvianolic acid for injection by ultra‐performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry

It is an essential requirement to clarify the metabolites of traditional Chinese medicine (TCM) injections, which contain numerous ingredients, to assess their safe and effective use in clinic. Salvianolic acid for injection (SAFI), made from hydrophilic phenolic acids in Salvia miltiorrhiza Bunge, has been widely used for the treatment of cerebrovascular diseases, but information on its metabolites in vivo is still lacking. In the present study, we aimed to holistically characterize the metabolites of the main active ingredients in rat plasma, bile, urine and feces following intravenous administration of SAFI. An ultra‐performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) method was developed. Combining information on retention behaviors, multistage mass spectra and literature data, a total of eight prototypes and 52 metabolites were tentatively characterized. Metabolites originated from rosmarinic acid and salvianolic acid B comprised the majority of identified compounds. Meanwhile, four metabolites derived from salvianolic acid D and five from salvianolic acid B are reported for the first time. This study revealed that methylation, sulfation and glucuronidation were the major metabolic pathways of phenolic acids in SAFI in vivo. Furthermore, the developed UPLC/Q‐TOF‐MS method could also benefit the metabolic investigation of extracts and preparations in TCM with hydrophilic ingredients. 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 major metabolites of quinocetone in swine urine using ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Quinocetone (QCT), 3‐methyl‐2‐cinnamoylquinoxaline‐1,4‐dioxide, is a quinoxaline‐N,N‐dioxide used in veterinary medicine as a feed additive. QCT is broadly used in China to promote animal growth, but few studies have been performed to reveal the metabolism of QCT in animals until now. In the present study, the metabolites of QCT in swine urine were investigated using ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight mass spectrometry (UPLC/ESI‐QTOF‐MS). Multiple scans of metabolites in MS and MS/MS modes and accurate mass measurements were performed simultaneously through data‐dependent acquisition. Most measured mass errors were less than ±5 mDa for both protonated molecules and product ions using external mass calibration. The structures of metabolites and their product ions were easily and reliably characterized based on the accurate MS² spectra and known structure of QCT. As expected, extensive metabolism was observed in swine urine. Thirty‐one metabolites were identified in swine urine, most of which were reported for the first time. The results reveal that the N‐O group reduction at position 1 and the hydroxylation reaction occurring at the methyl group, the side chain or on the benzene ring are the main metabolic pathways of quinocetone in swine urine. There was abundant production of 1‐desoxyquinocetone and hydroxylation metabolites of 1‐desoxyquinocetone. The proposed metabolic pathway of quinocetone in vivo can be expected to play a key role in food safety evaluations. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of isoquercitrin metabolites produced by human intestinal bacteria using UPLC‐Q‐TOF/MS

In this paper, ultraperformance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS) and the MetaboLynx? software combined with mass defect filtering were applied to identity the metabolites of isoquercitrin using an intestinal mixture of bacteria and 96 isolated strains from human feces. The human incubated samples collected for 72 h in the anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF/MS within 10 min. The parent compound and five metabolites were identified by eight isolated strains, including Bacillus sp. 17, Veillonella sp. 23 and 32 and Bacteroides sp. 40, 41, 56, 75 and 88 in vitro. The results indicate that quercetin, acetylated isoquercitrin, dehydroxylated isoquercitrin, hydroxylated quercetin and hydroxymethylated quercetin are the major metabolites of isoquercitrin. Furthermore, a possible metabolic pathway for the biotransformation of isoquercitrin was established in intestinal flora. This study will be helpful for understanding the metabolic route of isoquercitrin and the role of different intestinal bacteria in the metabolism of natural compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of astilbin metabolites produced by human intestinal bacteria using UPLC‐Q‐TOF/MS

Astilbin, mainly isolated from a commonly used herbal medicine, Smilax glabra Roxb (SGR), exhibits a variety of pharmacological activities and biological effects. It is metabolized by intestinal bacteria after oral administration which leads to the variation of ethnopharmacological profile of this traditional medicine. However, little is known on the interactions of this active compound with intestinal bacteria, which would be very helpful in unravelling how SGR works. In this study, different pure bacteria from human feces were isolated and were used to investigate their conversion capability of astilbin. Ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS) technique combined with Metabolynx^TM software was used to analyze astilbin and its metabolites. The parent compound and two metabolites (quercetin and eriodictyol) were detected in the isolated bacterial samples compared with blank samples. Quercetin was present in Enterococcus sp. 8B, 8–2 and 9–2 samples. Eriodictyol was only identified in Enterococcus sp. 8B sample. The metabolic routes and metabolites of astilbin produced by the different intestinal bacteria are reported for the first time. This will be useful for the investigation of the pharmacokinetic study of astilbin in vivo and the role of different intestinal bacteria in the metabolism of natural compounds. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic profile and pharmacokinetics of polyphyllin I,an anticancer candidate,in rats by UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS

Polyphyllin I (PPI), a natural steroidal saponin originating from rihzome of Paris polyphylla , is a potential anticancer candidate. Previous pharmacokinetics study showed that the oral bioavailability of PPI was very low, which suggested that certain amount of PPI might be metabolized in vivo . However, to date, information regarding the final metabolic fates of PPI is very limited. In this study, metabolites of PPI and their pharmacokinetics in rats were investigated using UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS. A total of seven putative metabolites, including six phase I and one phase II metabolites, were detected and identified with three exact structures by comparison with authentic standards for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. The pharmacokinetics of prosapogenin A, trillin and diosgenin, three deglycosylation metabolites of PPI with definite anticancer effects, were further studied, which suggested that the metabolites underwent a prolonged absorption and slower elimination after intragastric administration of PPI at the dose of 500 mg/kg. This study provides valuable and new information on the metabolic fate of PPI, which will be helpful in further understanding its mechanism of action.     

Characterization of metabolites in rat plasma after intravenous administration of salvianolic acid A by liquid chromatography/time‐of‐flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

Salvianolic acid A (SalA) is one of the main active constituents in Salvia miltiorrhiza (Danshen). Although the pharmacokinetics of SalA in rats after intravenous (i.v.) administration of Danshen injection has been reported, the information relevant to the metabolites of SalA in vivo is absent so far. In this study, by means of liquid chromatography with time‐of‐flight mass spectrometry (LC/TOFMS) and liquid chromatography with ion trap mass spectrometry (LC/MSⁿ) techniques, the unknown metabolites of SalA in rat plasma after i.v. administration of the purified SalA at the dose of 20 mg/kg body weight were identified. A liquid‐liquid extraction method was established to separate the metabolites from the plasma and the chromatographic separations were performed on a Xterra MS C₁₈ column (100 mm × 4.6 mm i.d., 3.5 µm) with acetonitrile/methanol/water/formic acid (20.5:19.5:64: 0.05, v/v/v/v) as the mobile phase at a constant flow rate of 0.2 mL/min. Based on the data obtained from the LC/TOFMS determination (the total ion chromatograms, MS spectra and extracted ion chromatograms), in combination with the characteristic fragment ions acquired from the LC/MSⁿ determination, five metabolites were identified as SalA‐monoglucuronide, monomethyl‐SalA‐monoglucuronide, mono‐methyl‐SalA, dimethyl‐SalA and dimethyl‐SalA‐monoglucuronide, and the possible chemical structures were deduced. The results indicated that SalA might mainly undergo two metabolic pathways in vivo in rats, which were methylation and glucuronidation. The present studies have laid a solid foundation for the metabolic mechanism of SalA in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of the absorbed components and their metabolites of Tianma‐Gouteng granule in rat plasma and bile using ultra‐high‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

Tianma‐Gouteng granule (TGG), a Chinese herbal formula preparation, is clinically used for the treatment of cardio‐cerebrovascular diseases such as hypertension, cerebral ischaemia, acute ischaemic stroke and Parkinson's disease. Although few reports have been published concerning the absorbed prototype components of TGG, the possible metabolic pathways of TGG in vivo remain largely unclear. In this study, a method using UPLC–Q/TOF MS was established for the detection and identification of the absorbed prototype components and related metabolites in rat plasma and bile after oral administration of TGG at high and normal clinical dosages. A total of 68 components were identified or tentatively identified in plasma and bile samples, including absorbed prototypes and their metabolites. The major absorbed components were gastrodin, isorhynchophylline, rhynchophylline, isocorynoxeine, corynoxeine, geissoschizine methyl ether baicalin, baicalein, wogonoside, wogonin, geniposidic acid, leonurine, 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐d ‐glucoside and emodin. The main metabolic pathways of these components involved phase I (isomerization, hydrolysis and reduction) and phase II (glucuronidation and sulfation) reaction, and the phase II biotransformation pathway was predominant. The present study provides rich information on the in vivo absorption and metabolism of TGG, and the results will be helpful for further studies on the pharmacokinetics and pharmacodynamics of TGG.     

Metabolism studies on prim‐O‐glucosylcimifugin and cimifugin in human liver microsomes by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Prim‐O‐glucosylcimifugin (PGCN) and cimifugin (CN) are major constituents of Radix Saposhnikoviae that have antipyretic, analgesic and anti‐inflammatory pharmacological activities. However, there were few reports with respect to the metabolism of PGCN and CN in vitro. In this paper, we describe a strategy using ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) for fast analysis of the metabolic profile of PGCN and CN in human liver microsomes. In total, five phase I metabolites of PGCN, seven phase I metabolites and two phase II metabolites of CN were identified in the incubation of human liver microsomes. The results revealed that the main phase I metabolic pathways of PGCN were hydroxylation and hydrolysis reactions. The phase I metabolic pathways of CN were found to be hydroxylation, demethylation and dehydrogenation. Meanwhile, the results indicated that O‐glucuronidation was the major metabolic pathway of CN in phase II metabolism. The specific UDP‐glucuronosyltransferase (UGT) enzymes responsible for CN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A9, UGT2B4 and UGT2B7 might play major roles in the glucuronidation of CN. Overall, this study may be useful for the investigation of metabolic mechanism of PGCN and CN, and it can provide reference and evidence for further pharmacodynamic experiments. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

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.     

Identification of metabolites of isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate (IDHP) is an investigational new drug having the capacity for treating ailments in the cardiovascular and cerebrovascular system. In this work, a rapid and sensitive method using high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐ESI‐Q‐TOF‐MS) was developed to reveal the metabolic profile of IDHP in rats after oral administration. The method involved pretreatment of the samples by formic acid–methanol solution (v/v, 5:95), chromatographic separation by an Agilent Eclipse XDB‐C₁₈ column (150 × 4.6 mm i.dx., 5 μm) and online identification of the metabolites by Q‐TOF‐MS equipped with electrospray ionizer. A total of 16 metabolites from IDHP, including four phase I metabolites and 12 phase II metabolites, were detected and tentatively identified from rat plasma, urine and feces. Among these metabolites, Danshensu (DSS), a hydrolysis product of IDHP, could be further transformed to 11 metabolites. These results indicated that DSS was the main metabolite of IDHP in rats and the major metabolic pathways of IDHP in vivo were hydrolysis, O‐methylation, sulfation, glucuronidation and reduction. The results also demonstrated that renal route was the main pathway of IDHP clearance in rat. The present study provided valuable information for better understanding the efficacy and safety of IDHP. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of bio‐active metabolites of gentiopicroside by UPLC/Q‐TOF MS and NMR

Gentiopicroside (GPS), the main bioactive component in Gentiana scabra Bge., has attracted our attention owing to its high bioactivity, especially the treatment of hepatobiliary disorders. The aglycone form of GPS, a typical secoiridoid glycoside, is considered to be more readily absorbed than its parent drug. This study aimed to identify and characterize the metabolites after GPS incubated with β‐glucosidase in buffer solution at 37°C. Samples of biotransformed solution were collected and analyzed by ultraperformance liquid chromatography (UPLC)/quadrupole–time‐of‐flight mass spectrometry (Q‐TOF MS). A total of four metabolites were detected: two were isolated and elucidated by preparative‐HPLC and NMR techniques, and one of those four is reported for the first time. The mass spectral fragmentation pattern and accurate masses of metabolites were established on the basis of UPLC/Q‐TOF MS analysis. Structure elucidation of metabolites was achieved by comparing their fragmentation pattern with that of the parent drug. A fairly possible metabolic pathway of GPS by β‐glucosidase was proposed. The hepatoprotective activities of metabolites M1 and M2 were investigated and the results showed that their hepatoprotective activities were higher than that of parent drug. Our results provided a meaningful basis for discovering lead compounds from biotransformation related to G. scabra Bge. in traditional Chinese medicine. Copyright © 2013 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.     

In vivo metabolism study of bergenin in rats by HPLC‐QTOF mass spectrometry

Bergenin is the major component of Ardisia creanta sims and Rodgersia sambucifolia hemsl with many biological activities. Although bergenin has been used to treat human diseases in China for man years, there is no report regarding its metabolism. This is the first report to separate and identify the metabolites of bergenin in vivo. In the study, HPLC/Q‐TOF‐MS/MS was used to investigate the metabolites of bergenin in vivo by analyzing the rat body fluid and feces samples. Three metabolites of bergenin were finally identified by the TIC chromatograms, and the structures were also confirmed by their MS² spectra. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of metabolites of sweroside in rat urine using ultra‐high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry and NMR spectroscopy

Sweroside, a major active iridoid in Swertia pseudochinensis Hara, is recognized as an effective agent in the treatment of liver injury. Based on previous reports, the relatively short half‐life (64 min) and poor bioavailability (approximately 0.31%) in rats suggested that not only sweroside itself but also its metabolites could be responsible for the observed hepato‐protective effect. However, few studies have been carried out on the metabolism of sweroside. Therefore, the present study aimed at identifying the metabolites of sweroside in rat urine after a single oral dose (100 mg/kg). With ultra‐high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UHPLC/Q‐TOF‐MS), the metabolic profile revealed 11 metabolites in rat urine, including phase I, phase II and aglycone‐related products. The chemical structures of metabolites were proposed based on accurate mass measurements of protonated or deprotonated molecules and their fragmentation patterns. Our findings showed that the aglycone of sweroside (M05) and its glucuronide conjugate (M06) were principal circulating metabolites in rats. While several other metabolic transformations, occurring via reduction, N‐heterocyclization and N‐acetylation after deglycosylation, were also observed. Two metabolites (M05 and M06) were isolated from the rat urine for structural elucidation and identifcation of reaction sites. Both M05 and M06 were characterized by ¹H, ¹³C and two‐dimensional nuclear magnetic resonance (NMR) spectroscopy. UHPLC/Q‐TOF‐MS analysis has provided an important analytical platform to gather metabolic profile of sweroside. Copyright © 2014 John Wiley & Sons, Ltd.