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.     

Studies on the metabolism and toxicological detection of glaucine,an isoquinoline alkaloid from Glaucium flavum (Papaveraceae), in rat urine using GC‐MS,LC‐MSn and LC‐high‐resolution MSn

Glaucine ((S)‐5,6,6a,7‐tetrahydro‐1,2,9,10‐tetramethoxy‐6‐methyl‐4H‐dibenzo [de,g]quinoline) is an isoquinoline alkaloid and main component of Glaucium flavum (Papaveraceae). It was described to be consumed as recreational drug alone or in combination with other drugs. Besides this, glaucine is used as therapeutic drug in Bulgaria and other countries as cough suppressant. Currently, there are no data available concerning metabolism and toxicological analysis of glaucine. To study both, glaucine was orally administered to Wistar rats and urine was collected. For metabolism studies, work‐up of urine samples consisted of protein precipitation or enzymatic cleavage followed by solid‐phase extraction. Samples were afterwards measured by liquid chromatography (LC) coupled to low or high‐resolution mass spectrometry (HR‐MS). The phase I and II metabolites were identified by detailed interpretation of the corresponding fragmentations, which were further confirmed by determination of their elemental composition using HR‐MS. From these data, the following metabolic pathways could be proposed: O‐demethylation at position 2, 9 and 10, N‐demethylation, hydroxylation, N‐oxidation and combinations of them as well as glucuronidation and/or sulfation of the phenolic metabolites. For monitoring a glaucine intake in case of abuse or poisoning, the O‐ and N‐demethylated metabolites were the main targets for the gas chromatography‐MS and LC‐MSⁿ screening approaches described by the authors. Both allowed confirming an intake of glaucine in rat urine after a dose of 2 mg/kg body mass corresponding to a common abuser's dose. Copyright © 2013 John Wiley & Sons, Ltd.     

Metabolic profiling of quercetin in rats using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

Quercetin, a kind of major flavonoid found in many traditional chinese medicines, is an effective substance for treatments such as lowering blood lipids. However, the studies on quercetin have been mainly focused on its pharmacological effect; the treatment of diseases on a material basis, particularly the metabolites derived from quercetin in vivo , has not been evaluated. In this study, we determined the levels, distributions and types of quercetin's metabolites in plasma, urine, feces and bile of rats after a single oral administration of quercetin at a dose of 80 mg/kg, using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS). A total of 36 metabolites of quercetin were identified, including 11 metabolites in plasma, 34 metabolites in urine, 12 metabolites in feces and 21 metabolites in bile. The results showed that phase I metabolites were reduction metabolites and phase II metabolites mainly included glucuronidation, sulfation and methylation metabolites. These results provide important information on the metabolism of quercetin, which will be helpful for its further development and utilization.     

Identification of circulatory and excretory metabolites of meisoindigo in rat plasma,urine and feces by high‐performance liquid chromatography coupled with positive electrospray ionization tandem mass spectrometry

Meisoindigo has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. However, information relevant to in vivo metabolism of meisoindigo is absent so far. In this study, in vivo circulatory metabolites of meisoindigo in rat plasma, as well as excretory metabolites in rat urine and feces, were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Integration of multiple reaction monitoring with conventional metabolic profiling methodology was adopted to enable a more sensitive detection of in vivo metabolites. By comparing with the MS/MS spectra and retention times of the in vitro reduced metabolites, the major metabolites in rat plasma were proposed to form from 3,3′ double bond reduction, whereas the minor metabolites were formed from reduction followed by N‐demethylation, and reduction followed by phenyl mono‐oxidation. The major metabolites in the rat urine were proposed to form from reduction followed by phenyl mono‐oxidation, and its glucuronide conjugation and sulfate conjugation, whereas the minor metabolites were formed from 3,3′ double bond reduction, N‐demethylation, reduction followed by N‐demethylation, phenyl di‐oxidation, phenyl mono‐oxidation and its glucuronide conjugation and sulfate conjugation. The major metabolites in the rat feces were proposed to form from reduction followed by phenyl mono‐oxidation, whereas the minor metabolites were formed from reduction followed by N‐demethylation, and reduction followed by phenyl di‐oxidation. The phase I metabolic pathways showed a significant in vitro–in vivo correlation in rat. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of the major metabolites of (R)‐salbutamol in human urine,plasma and feces using ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

(R)‐Salbutamol is a selective β2‐adrenoreceptor agonist, which produces a short‐acting bronchodilator effect and is widely used for the treatment of respiratory diseases in humans. Drug metabolism and identification of the metabolites play an essential role in the evaluation of the overall efficacy and safety of the drugs in clinical practices. There are few reports on the identification of major metabolites of (R)‐salbutamol in humans, and the number of identified metabolites is very limited. In this research, a method of ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was developed for the discovery and identification of (R)‐salbutamol and its major metabolites in human biological samples. Totally, twelve metabolites of (R)‐salbutamol were found and identified and all the metabolites could be found in urine, one metabolite in plasma and two metabolites in feces. Among all the metabolites, eight metabolites have never been reported before. The results indicated that (R)‐salbutamol was mainly metabolized through isomerization, oxidation, reduction, glucuronidation, and sulfation pathways in vivo. The possible metabolic pathways of (R)‐salbutamol were subsequently presented in this study, which contribute to a better understanding of the metabolism of (R)‐salbutamol in humans.     

Metabolite profiling of l‐isocorypalmine in rat urine,plasma, and feces after oral administration using high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry

l ‐Isocorypalmine, an active alkaloid compound isolated from Rhizoma Corydalis yanhusuo, has been reported to possess biological activity for treating cocaine use disorder. A high‐performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry method was established for identification of the metabolites of l ‐isocorypalmine in urine, plasma and feces samples of rats after a single intragastric gavage of l ‐isocorypalmine at a dose of 15 mg/kg. As a result, a total of 21 metabolites (six phase ? metabolites and fifteen phase II metabolites) were detected and tentatively identified by mass spectrometry and fragment ions from tandem mass spectrometry spectra. All metabolites were present in the urine samples, nine metabolites were found in the plasma samples and three metabolites were found in the feces samples. Results indicated that metabolic pathways of l ‐isocorypalmine included oxidation, dehydrogenation, demethylation, sulfate conjugation, and glucuronide conjugation. In addition, glucuronidation was the major metabolic reaction. Results of this investigation could provide significant experimental basis for efficacy, safety and action mechanism of l ‐isocorypalmine, which will be advantageous to new drug development for treating cocaine addiction.     

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.     

Rapid characterization of the absorbed chemical constituents of Tangzhiqing formula following oral administration using UHPLC–Q‐TOF‐MS

Tangzhiqing formula, a Chinese herbal formula, is used for the treatment of type II diabetes and prediabetes. Although its effectiveness has been certified by clinical use, its absorbed chemical constituents are not comprehensively represented. Thence, in order to reveal potential bioactive components and metabolism of Tangzhiqing formula, an ultra‐high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry method was developed. A total of 86 absorbed components, including 38 prototype compounds and 48 metabolites, were identified in rat plasma, urine, and feces after oral administration of Tangzhiqing formula. This was the first systematic study on the chemical constituents and metabolic profiling of Tangzhiqing formula. The results indicated that alkaloids and flavonoids were main absorbed components, and glucuronidation and sulfation were the major metabolites. Moreover we concluded that alkaloids and flavonoids first underwent demethylation and hydrolysis reactions before biotransformed to phase II metabolites. This study provided valuable data for safety estimation of Tangzhiqing formula, which will be advantageous for clinical application.     

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.     

Integrated strategy based on high‐resolution mass spectrometry coupled with multiple data mining techniques for the metabolic profiling of Xanthoceras sorbifolia Bunge husks in rat plasma,urine, and feces

An integrated strategy based on high‐resolution mass spectrometry coupled with multiple data mining techniques was developed to screen the metabolites in rat biological fluids after the oral administration of Xanthoceras sorbifolia Bunge husks. Mass defect filtering, product ion filtering, and neutral loss filtering were applied to detect metabolites from the complex matrix. As a result, 55 metabolites were tentatively identified, among which 45 barrigenol‐type triterpenoid metabolites were detected in the feces, and six flavonoids and four coumarins metabolites were in the urine. Moreover, eight prototype constituents in plasma, 36 in urine and 23 in feces were also discovered. Due to the poor bioavailability of barrigenol type triterpenoids, most of them were metabolized by intestinal flora. Phase I metabolic reactions such as deglycosylation, oxidation, demethylation, dehydrogenation, and internal hydrolysis were supposed to be their principal metabolic pathways. Coumarins were found in all the biosamples, whereas flavonoids were mainly in the urine. Unlike the saponins, they were mainly metabolized through phase II metabolic reactions like glucuronidation and sulfonation, which made them eliminated more easily by urine. This work suggested the metabolic profile of X. sorbifolia husks for the first time, which will be very valuable for its further development.     

Identification of metabolites in human and rat urine after oral administration of Xiao‐Qing‐Long‐Tang granule using ultra high performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

Xiao‐Qing‐Long‐Tang is a traditional Chinese formula used for the treatment of cold syndrome, bronchitis, and nasal allergies for thousands of years. However, the in vivo integrated metabolism of its multiple components and the active chemical constituents of Xiao‐Qing‐Long‐Tang remain unknown. In this study, a method using ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry was established for the detection and identification of the metabolites in human and rat urine after oral administration of Xiao‐Qing‐Long‐Tang. A total of 19 compounds were detected or tentatively identified in human urine samples, including eight prototypes and 11 metabolites. Also, a total of 50 compounds were detected or tentatively identified in rat urine samples, including 15 prototypes and 35 metabolites detected with either a highly sensitive extracted ion chromatogram method or the MS^E determination using Mass Fragment software. Our results indicated that phase Ⅱ reactions (e.g. glucuronidation and sulfation) were the main metabolic pathways of flavones, while phase I reactions (e.g. demethylation and hydroxylation) were the major metabolic reaction for alkaloids, lignans, and ginger essential oil. This investigation provided important structural information on the metabolism of Xiao‐Qing‐Long‐Tang and provided evidence to obtain a more comprehensive metabolic profile.     

Characterization of forsythoside A metabolites in rats by a combination of UHPLC‐LTQ‐Orbitrap mass spectrometer with multiple data processing techniques

Forsythoside A (FTA), the main active constituent isolated from Fructus Forsythiae, has various biological functions including anti‐oxidant, anti‐viral and anti‐microbial activities. However, while research on FTA has been mainly focused on the treatment of diseases on a material basis, FTA metabolites in vivo have not been comprehensively evaluated. Here, a rapid and sensitive method using a UHPLC‐LTQ‐Orbitrap mass spectrometer with multiple data processing techniques including high‐resolution extracted ion chromatograms, multiple mass defect filters and diagnostic product ions was developed for the screening and identification of FTA metabolites in rats. As the result, a total of 43 metabolites were identified in biological samples including 42 metabolites in urine, 22 metabolites in plasma and 15 metabolites in feces. These results demonstrated that FTA underwent a series of in vivo metabolic reactions including methylation, dimethylation, sulfation, glucuronidation, diglucuronidation, cysteine conjugation and their composite reactions. The research enhanced our understanding of FTA metabolism and built a foundation for further toxicity and safety studies.     

Metabolism studies of casticin in rats using HPLC‐ESI‐MSn

Casticin (3′,5‐dihydroxy‐3, 4′,6,7‐tetramethoxyflavone) has been revealed to possess various kinds of pharmacological activities, including immunomodulatory, anti‐hyperprolactinemia, anti‐tumor and neuroprotetective activities. In order to gain an understanding of the biotransformation of casticin in vivo, a systematic method based on liquid chromatography–electrospray ionization tandem mass spectrometry (LC‐ESI‐MSⁿ) was developed to identify the metabolites of casticin in rats after oral administration of single dose of casticin at 200 mg/kg. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent drug, the parent compound and 25 metabolites were identified in rat plasma, urine and six selected tissues. This is the first systematic metabolism study of casticin in vivo. The results indicated that methylation, demethylation, glucuronidation and sulfation were the main biotransformation pathways of casticin in vivo. Copyright © 2012 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.     

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.     

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.     

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.     

Characterization and identification of the metabolites of Menthae Haplocalycis Herba water extracts in rat plasma,urine, and feces by ultra‐high performance liquid chromatography with linear ion trap‐Orbitrap mass spectrometry

Menthae Haplocalycis Herba has been utilized for food and medicinal purposes in China for thousands of years. It has various efficacies, including dispelling wind and heat and relieving sore throat. M. Haplocalycis Herba has been also widely used in food, cosmetics, spices, and other fields. Exploring the constituents and detecting the metabolites of M. Haplocalycis are of great significance to clarify the effective substances. However, the in vivo metabolites of M. Haplocalycis Herba water extract are still unclear. Herein, a sensitive and specific method, ultra‐high performance liquid chromatography with linear ion trap‐Orbitrap mass spectrometry, established in this assay was used to study the metabolism of M. Haplocalycis Herba water extract in rat plasma, urine, and feces. We characterized and identified 9, 50, and 34 metabolites in plasma, urine, and feces, respectively. Seven metabolic pathways, including phase Ⅰ (isomerization, demethylation, hydroxylation, and dehydration) and phase Ⅱ (sulfation and glucuronidation) were mainly involved in the metabolism. It is the first systematic study on the metabolism of M. Haplocalycis Herba water extract in vivo, which enrich current understanding of the metabolic behavior of M. Haplocalycis Herba water extract and provide a metabolic rationale for further in‐depth in vivo biotransformation and pharmacokinetic analysis.     

Screening and identification of metabolites of two kinds of main active ingredients and hepatotoxic pyrrolizidine alkaloids in rat after lavage Farfarae Flos extract by UHPLC‐Q‐TOF‐MS mass spectrometry

Farfarae Flos, the dried flower buds of Tussilago farfara L., is usually used to treat coughs, bronchitic and asthmatic conditions as an important traditional Chinese medicine. Tussilagone and methl butyric acid tussilagin ester are seen as representatives of two kinds of active substances. In addition, the pyrrolizidine alkaloids, mainly senkirkine and senecionine, present in the herb can be hepatoxic. In this study, a rapid and sensitive ultra‐high‐performance liquid chromatography coupled with hybrid triple quadrupole time‐of‐flight mass spectrometry method was successfully applied to identify the metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine. A total of 35, 37, 18 and nine metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine in rats were tentatively identified. Hydrolysis, oxidation, reduction and demethylation were the major metabolic reactions for tussilagone and methl butyric acid tussilagin ester. The main biotransformation routes of senkirkine and senecionine were identified as demethylation, N‐methylation, oxidation and reduction. This study is the first reported analysis and characterization of the metabolites and the proposed metabolic pathways might provide further understanding of the metabolic fate of the chemical constituents after oral administration of Farfarae Flos extract in vivo.     

In vivo metabolism study of (R)‐bambuterol in humans using ultra high performance liquid chromatography with tandem mass spectrometry

(R)‐Bambuterol, a selective β2‐adrenoceptor agonist, has been approved as a new drug for the treatment of asthma and chronic obstructive pulmonary disease by the China Food and Drug Administration and is currently under phase I clinical trials. In this study, a combined method based on ultra high performance liquid chromatography with triple quadrupole mass spectrometry and ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was employed for the identification of the major metabolites of (R)‐bambuterol in human plasma and urine after an oral dose of 10 mg. The metabolites were separated by gradient elution program and different sample preparation methods were compared. Totally, 12 metabolites of (R)‐bambuterol were identified, including four metabolites in plasma and all 12 metabolites in urine. Among these, four metabolites are reported for the first time. The possible metabolic pathways of (R)‐bambuterol were subsequently proposed. The results indicated that (R)‐bambuterol was metabolized via hydrolysis, demethylation, oxygenation, glucuronidation, and sulfation pathways in vivo. This study revealed that this combined method was accurate and sensitive to identify the possible metabolites and to better understand the metabolism of (R)‐bambuterol in vivo.