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.     

UHPLC‐Q‐TOF‐MS/MS‐based screening and characterization of metabolites of cnidilin in human liver microsomes

Cnidilin is an active natural furocoumarin ingredient originating from well‐known traditional Chinese medicine Radix Angelicae Dahuricae . In the present study, an efficient approach was developed for the screening and identification of cnidilin metabolites using ultra‐high‐performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry. In this approach, an on‐line data acquisition method multiple mass defect filter combined with dynamic background subtraction was developed to trace all probable metabolites. Based on this analytical strategy, a total of 24 metabolites of cnidilin were detected in human liver microsomal incubation samples and the metabolic pathways were proposed. The results indicated that oxidation was the main biotransformation route for cnidilin in human liver microsomes. In addition, the specific cytochrome P450 (CYP) enzymes involved in the metabolism of cnidilin were identified using chemical inhibition and CYP recombinant enzymes. The results showed that CYP1A2 and CYP3A4 might be the major enzymes involved in the metabolism of cnidilin in human liver microsomes. The relationship between cnidilin and the CYP450 enzymes could provide us a theoretical basis of the pharmacological mechanism.     

Metabolites study on 5‐O‐methylvisammioside in vivo and in vitro by ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry

5‐O‐Methylvisammioside is one of major chromones of Radix Saposhnikoviae possessing definite pharmacological activities, but there are few reports with respect to the metabolism of 5‐O‐methylvisammioside. In this work, metabolites in vivo were explored in male Sprague‐Dawley rats and in vitro investigated on rat intestinal bacteria incubation model and were identified by using ultra high performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry. An online data acquisition method based on a multiple mass defect filter and dynamic background subtraction was developed to trace all probable metabolites. As a result, 26 metabolites in vivo (including 18, 15, 10, and 10 in rat urine, faece, bile, and blood) and 7 metabolites in vitro were characterized, respectively. Additionally, the main metabolic pathways in vivo and in vitro, including deglycosylation, deglycosylation + demethylation, deglycosylation + oxidation, N‐acetylation, and sulfate conjugation, were summarized by calculating the relative content of each metabolite. The obtained results significantly enriched our knowledge about 5‐O‐methylvisammioside metabolism and will lead to a better understanding of its safety and efficacy.     

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.     

Studies on metabolism of total glucosides of paeony from Paeoniae Radix Alba in rats by UPLC‐Q‐TOF‐MS/MS

Total glucosides of paeony are the active constituents of Paeoniae Radix Alba. In this study, a novel strategy was proposed to find more metabolites and the differences between paeoniflorin, albiflorin and total glucosides of paeony (TGP). This strategy was characterized as follows: firstly, the animals were divided into three groups (paeoniflorin, albiflorin and TGP) to identify the source of TGP metabolites from paeoniflorin or albiflorin; secondly, a generic information‐dependent acquisition scan for the low‐level metabolites was triggered by the multiple mass defect filter and dynamic background subtraction; thirdly, the metabolites were identified with a combination of data‐processing methods including mass defect filtering, neutral loss filtering and product ion filtering; finally, a comparative study was used in the metabolism of paeoniflorin, albiflorin and TGP. Based on the strategy, 18 metabolites of TGP, 10 metabolites of paeoniflorin and 13 metabolites of albiflorin were identified respectively. The results indicated that the hydrolysis, conjugation reaction and oxidization were the major metabolic pathways, and the metabolic sites were the glycosidic linkage, the ester bond and the benzene ring. This study is first to explore the metabolism of TGP, and these findings enhance our understanding of the metabolism and the interactions of paeoniflrin and albiflorin in TGP. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Study of the in vitro metabolism of TJ0711 using ultra high performance liquid chromatography with quadrupole time‐of‐flight and ultra fast liquid chromatography with quadrupole linear ion trap mass spectrometry

TJ0711 (1‐[4‐(2‐methoxyethyl)phenoxy]‐3‐[2‐(2‐methoxyphenoxy)ethylamino]‐2‐propanol) is a novel β‐adrenoreceptor blocker with vasodilating activity. The aim of this study was to investigate the in vitro metabolic properties of TJ0711 from both qualitative and quantitative aspects using mouse, rat, dog, and human liver microsomes as well as rat hepatocytes. Two modern liquid chromatography with tandem mass spectrometry systems, ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry and ultra fast liquid chromatography with quadrupole linear ion trap mass spectrometry, were utilized for the analysis. To better characterize the metabolic pathways of TJ0711, two major metabolites were incubated under the same conditions as that for TJ0711. TJ0711 was extensively metabolized in vitro, and a total of 34 metabolites, including 19 phase I and 15 phase II metabolites, were identified. Similar metabolite profiles were observed among species, and demethylation, hydroxylation, carboxylic acid formation, and glucuronidation were proposed as the major metabolic routes. Significant interspecies differences were observed in the metabolic stability studies of TJ0711. Furthermore, gender differences were significant in mice, rats, and dogs, but were negligible in humans. The valuable information provided in this work will be useful in planning and interpreting further pharmacokinetic, in vivo metabolism and toxicological studies of this novel β‐blocker.     

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.     

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.     

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 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.     

Metabolic profile of phillyrin in rats obtained by UPLC‐Q‐TOF‐MS

Forsythia suspensa Vahl (Oleaceae) is an important original plant in traditional Chinese medicine. The air‐dried fruits of Forsythia suspensa have long been used to relieve respiratory symptoms. Phillyrin is one of the main chemical constituent of Forsythia suspensa. A clear understanding of the metabolism of phillyrin is very important in rational clinical use and pharmacological research. In this study, the metabolism of phillyrin in rat was investigated for the first time using an ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) method. Bile, urine and feces were collected from rats after single‐dose (10 mg/kg) orally administered phillyrin. Liquid–liquid extraction and ultrasonic extraction were used to prepare samples. UPLC‐Q‐TOF‐MS analysis of the phillyrin samples showed that phillyrin was converted to a major metabolite, M26, which underwent deglucosidation, further dehydration and desaturation. A total of 34 metabolites were detected including 30 phase I and four phase II metabolites. The conjugation types and structure skeletons of the metabolites were preliminarily determined. Moreover, 28 new metabolites were reported for the first time. The main biotransformation route of phillyrin was identified as hydrolysis, oxidation and sulfation. These findings enhance our understanding of the metabolism and the real active structures of phillyrin. Copyright © 2015 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.     

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.     

In vivo metabolism study of vaccarin in rat using HPLC‐LTQ‐MSn

In order to illustrate the main biotransformation pathways of vaccarin in vivo, metabolites of vaccarin in rats were identified using a specific and sensitive high‐performance liquid chromatography–electrospray ionization linear ion trap mass spectrometry (LTQ XL?) method. The rats were administered a single dose (200 mg/kg) of vaccarin by oral gavage. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent drug, the parent compound and six metabolites were found in rat urine after oral administration of vaccarin. The parent compound and five metabolites were detected in rat plasma. In heart, liver and kidney samples, respectively, one, four and three metabolites were identified, in addition to the parent compound. Three metabolites, but no trace of parent drug, were found in the rat feces. This is the first systematic metabolism study of vaccarin in vivo. The biotransformation pathways of vaccarin involved methylation, hydroxylation, glycosylation and deglycosylation. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of absorbed constituents and metabolites in rat plasma after oral administration of Shen‐Song‐Yang‐Xin using ultra‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

In this study, a rapid and sensitive method by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry, and Metabolynx^TM software with mass defect filter technique was developed for screening and identification of the metabolites in rat plasma after oral administration of Shen‐Song‐Yang‐Xin capsule (SSYX). A total of 92 SSYX‐related xenobiotics were identified or characterized, including 45 prototypes and 47 metabolites. The results indicated that the absorbed constituents and metabolites mainly came from benzocyclooctadiene lignans, tanshinones, isoquinoline alkaloids and triterpenic acids, while phase I reactions (e.g. hydrogenation, hydroxylation, demethylation) and phase II reaction (glucuronidation) were the main metabolic pathways of these ingredients in SSYX. This is the first study on metabolic profiling of SSYX in rat plasma after oral administration. Furthermore, these findings provide useful information on the potential bioactive compounds, and enhance our understanding of the action mechanism of SSYX. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of metabolites of Danggui Buxue Tang in rat urine by liquid chromatography coupled with electrospray ionization time‐of‐flight mass spectrometry

A method coupling liquid chromatography with electrospray ionization time‐of‐flight mass spectrometry (LC/ESI‐TOF/MS) has been developed for rapid and sensitive analysis of rat urinary metabolite profile of Danggui Buxue Tang (DBT), a well‐known Chinese herbal formula. After oral administration of DBT, urine samples were collected during 0–24 h, and then pretreated by solid‐phase extraction. A total of 68 compounds including 13 parent compounds and 55 metabolites were detected in the drug‐containing urines compared with blank urines. The total analytical time was less than 20 min. Metabolites of DBT were identified using dynamic adjustment of the fragmentor voltage to produce structure‐relevant fragment ions. By using this approach, the mass accuracy of precursor and fragment ions was typically within ±5 ppm of the theoretical values, and enabled the identification of 43 metabolites including 27 isoflavanoid and 16 phthalide metabolites. Our results indicated that glucuronidation and sulfation were the major metabolic pathways of isoflavonoids, while glutathione conjugation, glucuronidation and sulfation were the main metabolic pathways of phthalides. No saponin‐related metabolites were detected. The results of the present study provided important structural information relating to the metabolism of DBT. Furthermore, this work demonstrated the potential of the LC/ESI‐TOF/MS approach for identification of metabolites from Chinese herbal medicines in urine. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolomics revealed the toxicity of cationic liposomes in HepG2 cells using UHPLC‐Q‐TOF/MS and multivariate data analysis

Cationic liposomes (CLs) are novel nonviral vectors widely used for delivering drugs or genes. However, applications of CLs are largely hampered by their cytotoxicity, partly because the potential mechanism underlying the cytotoxicity of CLs remains unclear. The aim of the present study was to explore the underlying mechanism of cytotoxicity induced by CLs on HepG2 cells. Differential metabolites were identified and quantified using ultra‐liquid chromatography quadrupole time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF/MS). The toxicity of CLs on HepG2 cells was evaluated by multivariate data analysis and statistics. Additionally, CCK‐8 assay, heatmap, pathway and co‐expression network were carried out to explore the relations between the metabolites and the pathways. The results showed a dose‐dependent toxic effect of CLs on HepG2 cells, with an IC₅₀ value of 119.9 μg/mL. Multivariate statistical analysis identified 42 potential metabolites between CLs exposure and control groups. Pathway analysis showed significant changes in pathways involving amino acid metabolism, energy metabolism, lipid metabolism and oxidative stress in the CLs exposure group vs the control group. Metabolites related to the above‐mentioned pathways included phenylalanine, methionine, creatine, oxalacetic acid, glutathione, oxidized glutathione, choline phosphate and several unsaturated fatty acids, indicating that cells were disturbed in amino acid metabolism, energy and lipid supply when CLs exposure‐induced injury occurred. It is concluded that CLs may induce cytotoxicity by enhancing reactive oxygen species in vitro , affect the normal process of energy metabolism, disturb several vital signaling pathways and finally induce cell death.     

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.     

The profiling and identification of the metabolites of 8‐prenylkaempferol and a study on their distribution in rats by high‐performance liquid chromatography with diode array detection combined with electrospray ionization ion trap time‐of‐flight multistage mass spectrometry

8‐Prenylkaempferol is a prenylflavonoid that has various bioactivities and benefits for human health. A high‐performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time‐of‐flight multistage mass spectrometry (HPLC‐DAD‐ESI‐IT‐TOF‐MSⁿ) method was established to profile and identify the metabolites of 8‐prenylkaempferol in rat in vivo and in vitro, and to study the distribution of these metabolites in rats for the first time. A total of 38 metabolites were detected and tentatively identified, 30 of which were identified as new compounds. The new in vivo metabolic reactions in rats of prenylflavonoids of isomerization, polymerization, sulfation, amino acid conjugation, vitamin C conjugation and other known metabolic reactions were found in the metabolism of 8‐prenylkaempferol. The numbers of detected metabolites in feces, urine, plasma, small intestine, stomach, kidneys, liver, heart, lungs, spleen and hepatic S9 fraction were 31, 19, 1, 20, 13, 8, 7, 3, 3, 1 and 11, respectively. This indicated that small intestine and stomach were the major organs in which the 8‐prenylkaempferol metabolites were distributed. Furthermore, 16 metabolites were determined to have bioactivities based on the literature and ‘PharmMapper’ analysis. These findings are useful for better comprehension of the effective forms, target organs and pharmacological actions of 8‐prenylkaempferol. Moreover, they provide a reference for the study of the metabolism and distribution of prenylflavonoid aglycone compounds. Copyright © 2015 John Wiley & Sons, Ltd.