Identification of metabolites of Ginkgolide B in vivo and in vitro using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

Ginkgolide B is a dietary diterpene with multiple pharmacological activities. However, current research on ginkgolide B is not comprehensive. The current study analyzed the metabolic profile of ginkgolide B in vivo and in vitro using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. To detect and identify the different metabolites in ginkgolide B, a novel data processing method was used as an assistant tool. A total of 53 different metabolites of ginkgolide B (38 phase I metabolites and 15 phase II metabolites) were detected relative to blank samples. The biotransformation route of ginkgolide B was identified as oxidation, dehydroxylation, hydrogenation, decarbonylation, demethylation, sulfate conjugation, glucose conjugation, methylation, and acetylation. The current study demonstrated a method for rapidly detecting and identifying metabolites and provided useful information to further characterize the pharmacology and mechanism of ginkgolide B. A method for the analysis of other diterpene metabolic components in vivo and in vitro was also established.

UHPLC-Q-TOF-MS/MS method based on four-step strategy for metabolites of hinokiflavone in vivo and in vitro.

Hinokiflavone (HF), belonging to biflavonoids, possesses excellent pharmacological activities, including anti-inflammatory, antioxidant and antitumor activity. Nevertheless, its metabolism in vivo (rats) and in vitro (rat liver microsomes and intestinal flora) is presently not characterized. In this study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) based on four-step strategy was a rapid method for the detection of HF metabolites. A total of 41 metabolites in vivo, 49 metabolites in vitro were characterized. It also verified that intestinal tract exceeds the liver in the biotransformation of HF. More significant, the main metabolic pathways for HF were mainly bio-transformed to various mono-flavone resulting from the rupture of connective CO bonds, which exhibited a large distinction with other biflavones. Noteworthily, glutamine conjugation and glycine conjugation were considered as unique metabolic pathways of HF. The information obtained from this study contributes to better understanding of pharmacological mechanism of HF.

Combining subsidence theory and slope stability analysis method for building damage assessment in mountainous mining subsidence regions.

Ground subsidence and surface cracks caused by coal mining are typical man-made geological hazards that can severely damage the ecological environment and buildings. In China, within the theme of sustained and stable development, accurate assessment of mining-related building damage is paramount in order to address the contradiction between coal mining enterprises and building owners. Previous research in China focused mainly on the mining areas of plains, and only a few studies have considered building damage caused by intensive mining in mountainous areas. First, based on field investigation, this study located ground surface cracks and assessed the damage to buildings in the village of Nanyetou in Shanxi Province (China) attributable to the exploitation of the 15110 working face of the Baiyangling coal mine. Second, based on the mining subsidence law and boundary angle, the surface influenced boundary caused by underground mining was determined. However, as the existing subsidence theory cannot adequately explain the phenomenon of building damage, the damage was investigated from the perspective of slope stability analysis, and the slope safety factor before and after working face mining were calculated using the Janbu method. The analytical results showed that slope instability due to a decrease of the safety factor because of the coal mining activity was the principal reason for damage to the village buildings, a finding that was confirmed by field survey and InSAR monitoring displacement. The results of this study could provide guidance and reference for the assessment of building damage caused by underground mining in mountain areas.

Ultrahigh-performance liquid chromatography coupled with triple quadrupole and time-of-flight mass spectrometry for the screening and identification of the main flavonoids and their metabolites in rats after oral administration of Cirsium japonicum DC. extract.

RATIONALE: Cirsium japonicum DC., a traditional Chinese medicine, has been shown to have anti-haemorrhagic and anti-tumour effects. Pharmacological studies have demonstrated that this curative effect may be related to flavonoids. The present work aimed to screen and identify the main flavonoids and their corresponding metabolites in rats after oral administration of Cirsium japonicum DC. extract. METHODS: A rapid and simple method based on ultrahigh-performance liquid chromatography coupled with triple quadrupole and time-of-flight mass spectrometry (UHPLC/QTOF-MS) was developed for the identification of the primary absorbing components and metabolites of the principal flavonoids. The absorbing components were first characterized, followed by the selection of representative constituents. In this study, the main flavonoids, pectolinarin, linarin and pectolinarigenin, were selected as templates to identify possible metabolites. RESULTS: A total of 27 metabolites were detected in rat blood, urine and bile samples. A hydrolysis reaction was the first step for pectolinarin and linarin, followed by oxidation and reduction reactions. However, phase II metabolites for pectolinarin and linarin were not detected. The primary biotransformation routes of pectolinarigenin were identified as oxidation, reduction, hydrolysis, and glucuronide and glucose conjugation. CONCLUSIONS: The metabolic pathways of pectolinarin, linarin and pectolinarigenin were summarized. This study not only proposed a practical strategy for rapidly screening and identifying metabolites but also provided useful information for further pharmacological studies and the design of new drugs based on Cirsium japonicum DC.

Simultaneous Determination of Six Coumarins in Rat Plasma and Metabolites Identification of Bergapten in Vitro and in Vivo.

Coumarins are abundant in Umbelliferae and Rutaceae plants possessing varied pharmacological activities. The objectives of this study are to develop and validate the method for determination of six coumarins in rat plasma by liquid chromatography coupled with tandem mass spectrometry (LC-MS) and identify the metabolites of bergapten by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), respectively. Data-dependent acquisition mode (DDA) was applied to trigger enhanced product ion (EPI) scans by analyzing multiple reaction monitoring (MRM) signals. An efficient data processing method "key product ions (KPIs)" was used for rapid detection and identification of metabolites as an assistant tool. The time to reach the maximum plasma concentration ( Tmax) for the six compounds ranged from 1 to 6 h. A total of 24 metabolites of bergapten were detected in vitro and in vivo. The results could provide a basis for absorption and metabolism of coumarins.

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

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

Qualitative and Quantitative Analyses of Active Constituents in Trollius ledebourii.

Trollius ledebourii has been more involved in Mongolian medicine and is often used as a type of tea for heat-clearing and detoxifying in the populus. In this study, a rapid and sensitive method was established for the qualitative and quantitative analyses of the major constituents in T. ledebourii. Ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was developed for the identification of the multi-constituents in T. ledebourii. A total of 37 chemical constituents in T. ledebourii extract were unambiguously or tentatively identified, including 17 flavonoid glycosides, 6 flavones, 3 flavonols, 1 dihydroflavone, 8 phenolic acids, 1 amide and 1 triterpene. Pectolinarin, naringenin, isorhamnetin, diosmetin, protocatechuic acid, paeonol, caffeic acid and ferulic acid were first detected in T. ledebourii and the buttercup family. High-performance liquid chromatography-quadrupole ion trap tandem mass spectrometry was applied for the simultaneous determination of 11 compounds, which were either with high contents or strong bioactivities. Satisfactory linearity was achieved with a wide linear range and fine determination coefficient (r > 0.9987). The overall recoveries ranged from 98.07 to 101.2%, and the precision in terms of RSD was <0.74%. The results might provide the basis for quality control analysis of T. ledebourii.

Metabolism profiling of nevadensin in vitro and in vivo by UHPLC-Q-TOF-MS/MS.

Nevadensin is major constituents of Lysionotus pauciflorus Maxim. (Chinese name: Shidiaolan), which has a variety of pharmacological effects such as anti-mycobacterium tuberculosis activities, antitussive, anti-inflammatory and anti-hypertensive. In this paper, we investigated the metabolism of nevadensin in vitro and in vivo. A strategy was firstly developed to identify the metabolites of nevadensin by using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). An on-line data acquisition method a multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to trace all probable metabolites. Furthermore, some assistant tools, such as key fragment ions (KFI), were employed for compound hunting and identification. Based on the proposed method, 23 metabolites were structurally characterized in vivo including 16 phase I and 7 phase II metabolites, and 12 metabolites were detected in vitro containing 10 phase I and 2 phase II metabolites. The results indicated that oxidation, hydrolysis, demethylation, methylation, sulfate conjugation and glucuronide conjugation were main metabolic pathways of nevadensin. In a word, this study maybe can provide reference and valuable evidence for further investigation of the metabolic mechanism of nevadensin.

Metabolism studies on hydroxygenkwanin and genkwanin in human liver microsomes by UHPLC-Q-TOF-MS.

Hydroxygenkwanin (HYGN) and genkwanin (GN) are major constituents of Genkwa Flos for the treatment of edema, ascites, cough, asthma and cancer. This is a report about the investigation of the metabolic fate of HYGN and GN in human liver microsomes and the recombinant UDP-glucuronosyltransferase (UGT) enzymes by using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). An on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to trace all probable metabolites. Based on this analytical strategy, three phase I metabolites and seven glucuronide conjugation metabolites of HYGN, seven phase I metabolites and 12 glucuronide conjugation metabolites of GN were identified in the incubation samples of human liver microsomes. The results indicated that demethylation, hydroxylation and o-glucuronidation were main metabolic pathways of HYGN and GN. The specific UGT enzymes responsible for HYGN and GN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A3, UGT1A9, UGT1A10 and UGT2B7 might play major roles in the glucuronidation reactions. Overall, this study may be useful for the investigation of metabolic mechanism of HYGN and GN, and it can provide reference and evidence for further experiments.

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.

Nontargeted SWATH acquisition mode for metabolites identification of osthole in rats using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

Osthole (OST), 7-methoxy-8-isopentenoxycoumarin, is the characteristic constituent found in Cnidium monnieri (L.) Cuss. and possesses excellent pharmacological activities, including anticancer, anti-apoptosis and neuroprotection. In this study, a rapid and reliable method based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) and MetabolitePilot2.0™ software with principal component variable grouping (PCVG) filtering was developed to observe probable metabolites of OST firstly. The high resolution mass data were acquired by data-independent acquisition mode (DIA), i.e., sequential window acquisition of all theoretical fragmentation spectra (SWATH), which could significantly improved the hit rate of low-level and trace metabolites. A novel data processing method 'key product ions (KPIs)' were employed for metabolites rapid hunting and identification as an assistant tool. A total of 72 metabolites of OST were detected in vitro and in vivo, including 39 metabolites in rat liver microsomes (RLMs), 20 metabolites in plasma, 32 metabolites in bile, 32 metabolites in urine and 37 metabolites in feces. The results showed that mono-oxidation, demethylation, dehydrogenation, sulfate conjugation and glucuronide conjugation were major metabolic reactions of OST. More significant, oxydrolysis, 3,4-epoxide-aldehylation, phosphorylation, S-cysteine conjugation and N-acetylcysteine conjugation were considered as unique metabolic pathways of OST, and phosphorylation, S-cysteine conjugation and N-acetylcysteine conjugation reactions were characterized in rat biological samples for the first time. Preparation of active metabolites will be greatly helpful in elucidating the potential biological mechanism of OST, and the proposed metabolic pathways of it might provide further understanding of the safety and efficacy of simple coumarins.

Metabolites identificaion of two bioactive constituents in Trollius ledebourii in rats using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

Orientin and vitexin, 4'-hydroxyl-2-phenylchromen-4-one, are both major flavones derivatives found in Trollius ledebourii possessing definite pharmacological activities. In this study, in vitro metabolisms investigated on rat liver microsomes (RLMs) and in vivo metabolisms explored on Male Sprague Dawley rats of orientin and vitexin were tested, respectively. A systematic method based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was developed to characterize metabolites by means of electrospray ionization (ESI) mass spectrometry in positive ion mode. An on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to observe probable relevant metabolites. By comparison of chromatographic behaviors with reference substances, exact protonated ions, MS/MS fragment ions and relevant literature, a total of 12 metabolites of orientin and 23 metabolites of vitexin were detected, respectively, which suggested that orientin is more metabolically stable than vitexin. Oxidation, methylation, acetylation, reduction, loss of C6H10O5 and glucuronide conjugation were the major biotransformation routes of both of them in rats. More significant, glutamine conjugation, loss of CO and loss of CH2O were the unique metabolic pathways of vitexin compared with that of orientin for the first time. Besides, most metabolites were observed in rat urine and feces, implying that urine and feces were the active metabolic places for flavones. This is the first study on metabolisms of orientin and vitexin in vitro and in vivo simultaneously and the proposed metabolic pathways of them might provide further understanding of their pharmacological mechanisms and later study on their excretion.