Ultra-performance Liquid Chromatography Coupled With Quadrupole Time-of-flight Tandem Mass Spectrometry Research Articles

Chemical Characterization and Metabolic Profiling of the Compounds in the Chinese Herbal Formula Li Chang Decoction by UPLC-QTOF/MS.

Background Li Chang decoction (LCD), a Chinese medicine formula, is commonly used to treat ulcerative colitis (UC) in clinics. Purpose This study aimed to identify the major components in LCD and its prototype and metabolic components in rat biological samples. Methods The chemical constituents in LCD were identified by establishing a reliable ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF/MS) method. Afterwards, the rats were orally administered with LCD, and the biological samples (plasma, urine, and feces) were collected for further analyzing the effective compounds in the treatment of UC. Result A total of 104 compounds were discriminated in LCD, including 26 flavonoids, 20 organic acids, 20 saponins, 8 amino acids, 5 oligosaccharides, 5 tannins, 3 lignans, 2 alkaloids, and 15 others (nucleosides, glycosides, esters, etc.). About 50 prototype and 94 metabolic components of LCD were identified in biological samples. In total, 29 prototype components and 22 metabolic types were detected in plasma. About 27 prototypes and 96 metabolites were discriminated in urine, and 34 prototypes and 18 metabolites were identified in feces. Conclusion The flavonoids, organic acids, and saponins were the major compounds of LCD, and this study promotes the further pharmacokinetic and pharmacological evaluation of LCD.

Identification and Analysis of Chemical Constituents and Rat Serum Metabolites in Gushuling Using UPLC-Q-TOF/MS Coupled with Novel Informatics UNIFI Platform

Gushuling (GSL), a well-known hospital preparation composed of traditional Chinese medicine (TCM), has been widely used in the clinical treatment of osteoporosis (OP) for decades due to its remarkable therapeutic effect. However, the chemical constituents of GSL are still unclear so far, which limits the in-depth study of its pharmacodynamic material basis and further restricts its clinical application. In this study, we developed a strategy for qualitative analysis of the chemical constituents of GSL in vitro and in vivo. Based on the results of ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS) and the UNIFI informatics platform, the chemical constituents of GSL can be determined quickly and effectively. By comparing the retention time, accurate mass, and fragmentation spectrum of the compounds in GSL, a total of 93 compounds were identified or preliminarily identified, including flavonoids, terpenoids, phenylpropanoids, steroids, etc. Among them, nine compounds have been confirmed by standard substances, namely epimedin A, epimedin B, epimedin C, icariin, ecdysterone, calycosin, calycosin-7-glucoside, ononin, and ginsenoside Ro. Fragment patterns and characteristic ions of representative compounds with different chemical structure types were analyzed. At the same time, 20 prototype compounds and 42 metabolites were detected in rat serum. Oxidation, hydration, reduction, dehydration, glutathione S-conjugation, and acetylcysteine conjugation were the main transformation reactions of GSL in rat serum. In this research, the rapid method to characterize the in vitro and in vivo chemical constituents of GSL can not only be used for the standardization and quality control of GSL but also be helpful for further research on its pharmacodynamic material basis.

Chemical profiling of root bark extract from Oplopanax elatus and its in vitro biotransformation by human intestinal microbiota.

Oplopanax elatus (Nakai) Nakai, in the Araliaceae family, has been used in traditional Chinese medicine (TCM) to treat diseases as an adaptogen for thousands of years. This study established an ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) method to identify chemical components and biotransformation metabolites of root bark extract from O. elatus. A total of 18 compounds were characterized in O. elatus extract, and 62 metabolites by human intestinal microbiota were detected. Two polyynes, falcarindiol and oplopandiol were recognized as the main components of O. elatus, whose metabolites are further illustrated. Several metabolic pathways were proposed to generate the detected metabolites, including methylation, hydrogenation, demethylation, dehydroxylation, and hydroxylation. These findings indicated that intestinal microbiota might play an essential role in mediating the bioactivity of O. elatus.

Study on effect of "Trichosanthis Fructus-Allii Macrostemonis Bulbus" on atherosclerosis in ApoE~(-/-) mice based on liver metabonomics

In this study, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS)-based liver metabolomics approach was used to explore the mechanism of "Trichosanthis Fructus-Allii Macrostemonis Bulbus" in improving atherosclerosis(AS) of mice with apolipoprotein E gene knockout(ApoE~(-/-)). AS mouse model was induced by high-fat diet. The pathological and biochemical indexes such as the histopathological changes, body weight, liver weight, blood lipid level and inflammatory factors in the liver of mice were determined. The metabolic profiling of mice liver samples was performed with UPLC-Q-TOF-MS. Multiple statistical analysis methods including partial least squares discriminant analysis(PLS-DA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were employed to screen and identify biomarkers. The levels of related enzymes including LCAT, sPLA2, EPT1 and ACER1 were detected. The results showed that "Trichosanthis Fructus-Allii Macrostemonis Bulbus" significantly reduced the areas of aortic plaque and fat vacuoles of liver in AS mice and decreased the accumulation of lipid droplets and liver coefficient. "Trichosanthis Fructus-Allii Macrostemonis Bulbus" also regulated the levels of blood lipid and inflammatory injury in the liver. The metabolites of the control group, the model group and the "Trichosanthis Fructus-Allii Macrostemonis Bulbus" group could be distinguished significantly. Fifteen potential biomarkers related to AS were discovered and preliminarily identified, seven of which could be regulated by "Trichosanthis Fructus-Allii Macrostemonis Bulbus" in a trend of returning to normal. Metabolic pathway analysis screened out two major metabolic pathways. "Trichosanthis Fructus-Allii Macrostemonis Bulbus" obviously regulated the levels of LCAT, sPLA2, EPT1 and ACER1. It was inferred that "Trichosanthis Fructus-Allii Macrostemonis Bulbus" could play a major role in AS treatment by regulating glycerophospholipid and sphingolipid metabolism disorders in the liver, with the mechanism probably relating to the intervention of the expression of LCAT, sPLA2, EPT1 and ACER1.

Metabolic profiles of Jin‐hong tablets in rats by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry

Jin-hong tablets (JHTs), a well-known traditional Chinese patent medicine (TCPM), have been effectively used for the treatment of chronic superficial gastritis (CSG). The metabolic profile of TCPMs is performed to determine their bioactive components. In this study, a five-step strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and metabolynx™ software combined with mass defect filter technique was developed to delineate the metabolic profile of JHT in vivo. As a result, a total of 163 JHT-related xenobiotics (38 prototypes and 125 metabolites) were identified or tentatively characterized in rat biological samples, and the phase I and II metabolism processes mainly included demethylation, hydroxylation, sulfation, and glucuronidation. In addition, after oral administration of JHT, a large amount of alkaloid-related ingredients was detected in rat plasma samples, indicating that alkaloids may play an important role in the treatment of CSG with JHT. This study is beneficial for understanding the JHT's in vivo metabolic profiles and characteristics, which helps to reveal its in vivo effective components and provides a solid basis for further studies on its functional mechanism.

Identifying the molecular basis of Jinhong tablets against chronic superficial gastritis via chemical profile identification and symptom-guided network pharmacology analysis

Chronic superficial gastritis (CSG) is a common disease of the digestive system that possesses a serious pathogenesis. Jinhong tablet (JHT), a traditional Chinese medicine (TCM) prescription, exerts therapeutic effects against CSG. However, the molecular basis of its therapeutic effect has not been clarified. Herein, we employed ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) based chemical profile identification to determine the chemical components in JHT. Further, we applied network pharmacology to illustrate its molecular mechanisms. A total of 96 chemical constituents were identified in JHT, 31 of which were confirmed using reference standards. Based on the bioinformatics analysis using the symptom-guided pharmacological networks of “chi,” “blood,” “pain,” and “inflammation,” and target screening through the interaction probabilities between compounds and targets, matrix metalloproteinase 2 (MMP2), dopamine d2 receptor (DRD2), and Aldo-keto reductase family 1 member B1 (AKR1B1) were identified as key targets in the therapeutic effect exhibited by JHT against CSG. Moreover, according to the inhibitory activities presented in the literature and binding mode analysis, the structural types of alkaloids, flavonoids, organic acids, including chlorogenic acid (10), caffeic acid (13), (−)-corydalmine (33), (−)-isocorypalmine (36), isochlorogenic acid C (38), isochlorogenic acid A (41), quercetin-3-O-α-l-rhamnoside (42), isochlorogenic acid B (47), quercetin (63), and kaempferol (70) tended to show remarkable activities against CSG. Owing to the above findings, we systematically identified the chemical components of JHT and revealed its molecular mechanisms based on the symptoms associated with CSG.

Rapid identification of absorbed components and metabolites of Gandou decoction in rat plasma and liver by UPLC-Q-TOF-MSE

Gandou Decoction (GDD), a well-known traditional Chinese medicine prescription, has been widely used for decades in clinical practice to treat Wilson’s disease (WD) in China. However, due to lack of in vivo metabolism research, the absorbed components and metabolites of GDD have not been fully elucidated. In this study, a rapid and high-throughput ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MSE) was applied to rapidly identify prototypes and metabolites after oral administration of GDD. On this basis, the possible metabolic pathways of the main prototypes were proposed between normal and copper-laden rats. As a result, a total of 89 GDD-related xenobiotics were detected in normal dosed rats, including 83 (36 prototypes and 47 metabolites) in plasma and 52 (21 prototypes and 31 metabolites) in liver; a total of 77 GDD-related xenobiotics were detected in copper-laden dosed rats, including 68 (31 prototypes and 37 metabolites) in plasma and 42 (19 prototypes and 23 metabolites) in liver. Our findings showed that anthraquinones, alkaloids and protostane triterpenoids as well as a few saponins, flavonoids, tannins and curcuminoids were the main absorbed chemical components of GDD in rat plasma; anthraquinones, protostane triterpenoids and curcuminoids were the major components in rat liver. Glucuronidation and sulfation were deduced to be the predominant metabolic pathways of GDD. Methylation, acetylation, reduction, hydroxylation, demethylation and deglycosylation were often occurred in the metabolic process. Furthermore, the holistic metabolic profile of GDD revealed that copper-laden rats and normal rats had certain differences in drug absorption and metabolism. This study offered a solid basis for ascertaining bioactive components and action mechanism of GDD.

An animal research and a chemical composition analysis of a Chinese prescription for pulmonary fibrosis: Yangfei Huoxue Decoction

Ethnopharmacological evidencePulmonary fibrosis (PF) is a progressive disease characterized by the aberrant accumulation of fibrotic tissue in the lungs parenchyma, associated with significant morbidity. Few effective drugs have been developed to reverse PF or even halt the disease progression. Yangfei Huoxue Decoction (YHD), a Traditional Chinese Medicine, which consisted of Astragalus membranacus(AM), Glehnia littoralis(GL), Schisandra chinensis(SC), Salvia miltiorrhiza Bunge(SB), Reynoutria japonica(RJ), Ligusticum chuanxiong(LX), and Euonymus alatus(EA) , has been used in China for the treatment of PF for many years with remarkable efficacy.According to the clinic observation of the results, we conducted experiments on animals, the process of BLM-induced pulmonary fibrosis in rats was interfered by YHD, through the detection of pulmonary fibrosis rats’ blood cells and plasma, we selected the related molecules that may exert proinflammatory(IL-1β), promote angiogenesis(vascular endothelial growth factor ,VEGF).For further explicitly research, we should know what the chemical composition the prescription (YHD) contains and what the related bioactive components have.In accordance with in-house library and evaluating the characteristic MS fragmentation patterns, the schisandra chinensis methanol, lignin, flavonol, polyphenol, tanshinone, salvianolic acid, anthraquinone, ligustrazine, etc. had a retardant and inhibitory effect on the development and formation of pulmonary fibrosis. These results will aid in the quality control of YHD, as well as provide fundamental data for further pharmaco-mechanisms studies. Aim of the studyTo discover the pulmonary immune related bioactive components of YHD. Materials and methodsAnimal Experiment:144 SD rats, based on the principles of randomization divided into eight groups, Control group, bleomycin(BLM) group, BLM + dexamethasone(BLM + DXM) group, BLM + Yangfei(YF) group, BLM + Huoxue(HX) group, BLM + high-doseYHD(YHD-H) group, BLM + medium-doseYHD(YHD-M) group, and BLM + low-doseYHD(YHD-L) group, each group of 18 rats. After endotracheal administration of Bleomycin by tracheotomy, rats were sacrificed on day 7, day 14 and day 28, blood and plasma were taken at the same time. Respectively, the VEGF, an immune molecule associated with angiogenesis, and IL-1β in plasma were detected by ELISA at three time periods. Component testing: 100 g YHD were constituted of SB 15 g, LX 12 g, EA 10 g, RJ 15 g, AM 20 g, GL 20 g and SC 8 g. All herbs were obtained from Beijing Tong Ren Tang (Group) Co ltd. The voucher specimens were identified by Prof. Jiening Gong (Nanjing University of Chinese Medicine). YHD were extracted by sonication with 1 L ethanol/water (70:30, v/v) for two cycle (1 h per cycle) at room temperature. The combined extracts were filtered, condensed, and reconstituted with 50 mL methanol before analysis. Standard Cianidanol, Ferulic Acid, Polydatin, Calycosin 7-O-glucoside, Tanshinone IIA, Salvianolic acid B, Schizandrol A, and Isoimperatorin were prepared in methanol. After centrifuging at 20,000 rpm for 10 min, 4 μL supernatant was injected into the Ultra-Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight tandem mass spectrometry (UPLC/QTOF-MSE) combined with UNIFI informatics platform for analysis. ConclusionThe experiment results revealed that the vascularized VEGF, inflammatory factor expression of IL-1β was restrained by YHD.The UPLC/QTOF-MSE method, an automatic database screening platform and the characteristic MS fragmentation patterns have efficiently facilitated the post data process, so we test for the identification of major components in YHD by this technology, more than seven or more active ingredients, the results showed that YHD contained a total of 55 components, including 11 lignans, 12 flavonoids, 7 tanshinones, 9 organic acid, 5 polyphenols, 4 anthraquinones, 5 senkyunolides and 2 others. Based on this, we can ensure the discovery and analysis of biologically active compounds in YHD, as well as provide a reference for the quality evaluation. We expect the method presented here could be applied to other multi-component TCM formula. In addition, we can conduct more in-depth research, such as mechanism research, molecular detection, gene target and so on.

Rapid identification of chemical profile in Gandou decoction by UPLC-Q-TOF-MSE coupled with novel informatics UNIFI platform

Gandou decoction (GDD), a well-known traditional Chinese medicine (TCM) formula, has been widely used for decades to treat Wilson's disease (WD) in China due to its remarkable clinical effects. However, the chemical constituents of GDD still remain unclear because of their complexity. In this work, a reliable and sensitive strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MSE) and UNIFI informatics platform was applied to investigate the chemical components in GDD. In total, 96 compounds including anthraquinones, alkaloids, protostane triterpenoids, flavonoids, triterpenoid saponins, tannins, curcuminoids, etc, were identified or tentatively characterized from GDD by comparing their retention time, accurate mass within 5 ppm error and MSE fragmentation patterns. Among them, eleven compounds were confirmed unambiguously with reference standards. Representative compounds in different chemical structure types were analyzed in fragmentation patterns and characteristic ions. Moreover, to better understand the chemical contribution of individual herbs to the whole decoction, the corresponding each herb in GDD was also detected. This study developed a rapid method for characterizing the chemical constituents in GDD, which could not only be used for chemical standardization and quality control, but also be helpful for further research of GDD in vivo.

Mucosal immunomodulatory evaluation and chemical profile elucidation of a classical traditional Chinese formula, Bu-Zhong-Yi-Qi-Tang

Ethnopharmacological evidenceWith fast development and high pace life in modern society, autoimmune diseases like inflammatory bowel disease had become increasingly common. Bu-Zhong-Yi-Qi-Tang (BZYQT), a famous traditional Chinese medicine prescription (TCMP), has been used for 700 years mainly in Eastern Asia countries for the treatment of gastrointestinal and respiratory disorder, and weakness after serious diseases. These diseases were proved to be closely related to human immune system, among which, mucosal immune system is the largest immune system. So it is necessary to discover the mucosal immune related bioactive components of BZYQT. Aim of the studyTo evaluate the mucosal immunomodulatory bioactivity of BZYQT and ingredients. Materials and methodsPeyer's patches were collected from mice administrated orally with BZYQT, its related Octadecylsilane (ODS) fractions and polysaccharide part. Productions of several cytokines including IL-2, IL-4, IL-5, and IFN-γ from T lymphocytes were tested with enzyme linked immunosorbent assay (ELISA) by Peyer's patch cells ex vivo experiments. Chemical profile including low molecular part and polysaccharide part were investigated. Low molecular part of BZYQT and related ODS fractions were analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) based on LC-MS information from self-established compound library. exclusion chromatography, and chemical property has been analyzed. ResultThree-days’ administration of BZYQT enhanced productions of IL-4 and IFN-γ in T lymphocytes of Peyer's patches in addition to IL-2. Some hydrophobic low molecular weight fractions (30%, 70% and 100% MeOH ODS fraction), which were fractionated from BZYQT by ODS column chromatography, showed enhancing or suppressive effects on productions of IL-2, IL-4 or IL-5 in T lymphocytes of Peyer's patches after oral administration. Besides, 161 components from hydrophobic low molecular weight fractions of BZYQT were unequivocally identified or tentatively characterized by UPLC-Q/TOF-MS according to retention time behaviors and fragments, and most of them were flavonoids and saponins from Glycyrrhizae Radix, Citri Reticulatae Pericarpium, and Cimicifugae Rhizoma. Polysaccharide part was separated and purified both by anion-exchange and size. BZYQT also contained at least one neutral and three weakly or strongly acidic polysaccharides, and analysis of their chemical properties indicated that a neutral polysaccharide was glucan, and acidic polysaccharides possessed heteroglycan and pectic arabinogalactan features. Murine administration of polysaccharide fractions of BZYQT induced different changes on functions of T lymphocytes in Peyer's patches from hydrophobic low molecular weight fractions. By experiment using intranasally-immunized mice, BZYQT negatively regulated antibody response in lung as combinatorial actions of its low molecular weight ingredients and polysaccharides. ConclusionBZYQT contains several low and macromolecular weight ingredients, which affect to immune-function of T lymphocytes in Peyer's patches, and the formula expresses its regulative activity on lower respiratory immune system through combinatorial actions of these ingredients on immunocompetent cells in Peyer's patches.

Metabolites profile of Gualou Xiebai Baijiu decoction (a classical traditional Chinese medicine prescription) in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry

Gualou Xiebai Baijiu decoction (GLXB), a well-known classic traditional Chinese medicine prescription, has been widely used to treat coronary heart diseases for thousands of years in Eastern Asian countries due to its remarkable clinical effect. However, due to lack of in vivo metabolism research, the chemical components responsible for the therapeutic effects still remain unclear. In this work, a reliable “representative structure based homologous xenobiotics identification” (RSBHXI) strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) were applied to investigate the chemical components in GLXB extracts. As a result, 133 chemical components were characterized based on summarized fragmentation patterns, of which 41 components were confirmed unambiguously with authentic standards. Furthermore, a total of 138 GLXB-related xenobiotics were identified or tentatively characterized after oral administration of GLXB extracts. Moreover, to better understand the metabolic pathways of characteristic components in GLXB, metabolites profiles of five steroidal saponins and two flavonoids were performed, respectively. Since the metabolic pathways of five representative saponins had been finished in our previous study, we focused on the in vivo metabolism of two flavonoids. A total of 36 and 20 metabolites were detected in rat biological samples after oral administration of luteolin-7-O-β-D-glucopyranoside and rutin, respectively. The results indicated that dehydration, hydrolysis, hydroxylation, methylation, glucuronidation and sulfation were the main metabolic reactions, following the metabolic soft spots of GLXB-related flavonoids. Taken altogether, this study would be helpful for the further pharmacokinetics, pharmacological evaluation and quality control of GLXB.

Pharmacokinetic Investigation of Main Bioactive Components from Epimedium-Derived Flavonoids in Rabbit Serum by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry

Background: Total Epimedium-derived flavonoids (EF) were reported to be a group of major bioactive constituents present in genus Epimedium for the treatment of osteonecrosis. The present study aimed to investigate the pharmacokinetics of main bioactive components of EF in rabbit. Methods: A rapid and sensitive method for the simultaneous determination of icariin, icariside I, icariside II and icaritin in serum was established and validated by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS). Serum samples were pretreated with water-saturated ethyl acetate. Furthermore, sample separation was achieved on an Waters BEH C18 column (50 mm × 2.1 mm, 1.7 μm) by gradient elution with acetonitrile-water (both including 0.1% formic acid) as mobile phase. Finally, pharmacokinetics of EF in rabbit serum was investigated. Results: All calibration curves displayed excellent linearity (r2>0.99) within 1.2~24 ng/mL. The assay was specific, precise and accurate, as demonstrated with intra-run precisions not more than 12.6%, inter-run precisions less than 9.9% and accuracies between -4.4% and 9.3%. Meanwhile, the matrix effects, extraction recoveries and stabilities were all satisfactory. Moreover, this validated method was successfully applied to analyse the pharmacokinetic of EF in rabbit serum after oral dosage of 80 mg/kg. Conclusion: These results would enlarge our knowledge about main bioactive components of EF for the treatment of osteonecrosis in rabbit.

Identification, bioactivity evaluation and pharmacokinetics of multiple components in rat serum after oral administration of Xian-Ling-Gu-Bao capsule by ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

The Xian-Ling-Gu-Bao capsule (XLGB) is a famous traditional Chinese medicine prescription (TCMP), which has proven effective in osteoporosis treatment. However, due to the lack of a dynamic XLGB profile, the in vivo pharmacokinetics of multiple bioactive components within this medicine remains unknown. In the present study, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) identified a total of eighteen prototypes (using reference standards) in rat serum after oral administration of XLGB. These prototypes were subsequently evaluated to ascertain their effects on the proliferation and alkaline phosphatase activity of UMR106 cells and the adipogenesis of 3T3-L1 cells. Furthermore, a rapid and sensitive UPLC/Q-TOF-MS method was developed and validated for simultaneous quantitative analysis of 11 prototypes in rat serum. Chromatographic separation was achieved using a Waters Acquity BEH C18 column (2.1×100mm, 1.7μm) and linear gradient elution employing a mobile phase consisting of water and acetonitrile (both containing 0.1% formic acid). All calibration curves showed excellent linearity (r2>0.99) within the sampling ranges considered. The assay was accurate, precise and reproducible, as demonstrated by the obtained intra- and inter-day precisions (less than 12.3%) and accuracies (between -12.7% and 11.0%), and the matrix effects, extraction recoveries and stabilities were all satisfactory. Moreover, pharmacokinetic parameters were calculated from the plasma concentration-time data. Compared to single-compound dosing, significantly enhanced responses were obtained when several analytes were administered simultaneously, indicating possible drug-drug interactions among the complex ingredients of TCMP. This work provides an experimental baseline regarding the clinical applications and medicinal effectiveness of XLGB in the treatment of osteoporosis.

Metabolism of 20(S)-Ginsenoside Rg₂ by Rat Liver Microsomes: Bioactivation to SIRT1-Activating Metabolites.

20(S)-Ginsenoside Rg2 (1) has recently become a hot research topic due to its potent bioactivities and abundance in natural sources such as the roots, rhizomes and stems-leaves of Panax ginseng. However, due to the lack of studies on systematic metabolic profiles, the prospects for new drug development of 1 are still difficult to predict, which has become a huge obstacle for its safe clinical use. To solve this problem, investigation of the metabolic profiles of 1 in rat liver microsomes was first carried out. To identify metabolites, a strategy of combined analyses based on prepared metabolites by column chromatography and ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) was performed. As a result, four metabolites M1–M4, including a rare new compound named ginsenotransmetin A (M1), were isolated and the structures were confirmed by spectroscopic analyses. A series of metabolites of 1, MA–MG, were also tentatively identified by UPLC-Q-TOF/MS in rat liver microsomal incubate of 1. Partial metabolic pathways were proposed. Among them, 1 and its metabolites M1, M3 and M4 were discovered for the first time to be activators of SIRT1. The SIRT1 activating effects of the metabolite M1 was comparable to those of 1, while the most interesting SIRT1 activatory effects of M3 and M4 were higher than that of 1 and comparable with that of resveratrol, a positive SIRT1 activator. These results indicate that microsome-dependent metabolism may represent a bioactivation pathway for 1. This study is the first to report the metabolic profiles of 1 in vitro, and the results provide an experimental foundation to better understand the in vivo metabolic fate of 1.

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.

Identification of metabolites of PSORALEAE FRUCTUS in rats by ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry analysis

The fruit of Psoralea corylifolia (PSORALEAE FRUCTUS) is a traditional Chinese medicine (TCM), which has been used to prevent and treat vitiligo, psoriasis, and osteoporosis in China for thousands of years. Phytochemical investigation on PSORALEAE FRUCTUS, as well as some metabolism research focused on pharmacokinetics of several single compounds from this plant, has been reported. However, the effective material of PSORALEAE FRUCTUS is still unknown. In the present study, the metabolic fate of multiple components of PSORALEAE FRUCTUS in rats was investigated by ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS). Based on a three-step strategy, a total of 142 PSORALEAE FRUCTUS-related xenobiotics were identified or tentatively characterized in rat biofluids after oral administration of six representative single compounds and PSORALEAE FRUCTUS extract. All six different types of constituents of PSORALEAE FRUCTUS, including furocoumarin, coumestan, isoflavone, flavanone, chalcone and monoterpene phenol, could be absorbed into the circulation system. In addition, compared with the metabolism of six representative single compounds, different metabolic fate was observed after oral administration of PSORALEAE FRUCTUS extract, which indicated that the drug–drug interactions occurred when fed by multi-component herbal extract, and the investigations only focused on several main components were not sufficient to represent and reflect the overall efficacy of plants. The present study will be conducive to further pharmacological mechanism research on PSORALEAE FRUCTUS.

Metabolites profile of Xian-Ling-Gu-Bao capsule, a traditional Chinese medicine prescription, in rats by ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry analysis

Xian-Ling-Gu-Bao capsule (XLGB), a well-known traditional Chinese medicine prescription (TCMP), is widely used for the treatment of osteoporosis. However, due to lack of metabolism research, the effective material of XLGB is still unknown. It entails a huge obstacle for the clinical-safe medication administration and quality control of XLGB. To explore the metabolic fate of multiple components of XLGB, herein, we proposed a “representative structure based homologous xenobiotics identification” (RSBHXI) strategy based on ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) and mass defect filter (MDF) technique. A total of 147 XLGB-related xenobiotics were identified or tentatively characterized in rat biofluids after oral administration of XLGB, including 134 (57 prototype components and 77 metabolites) in plasma, 93 (37 prototype components and 56 metabolites) in urine and 118 (46 prototype components and 72 metabolites) in bile. Our results indicated that prenylated flavonol glycosides from Herba epimedii, prenylated flavonoids from Fructus psoraleae, saponins from Radix dipsaci and Rhizoma anemarrahenae, as well as tanshinones from Radix Salviae Miltiorrhizae were major absorbed chemical components of XLGB. Hydrolysis, glucuronidation and sulfation were major metabolic reactions of XLGB. As more xenobiotics were detected in bile than those in urine, it demonstrated that multiple components of XLGB underwent comprehensive hepatobiliary excretion. The present study expands our knowledge about the metabolism of XLGB which will be conducive to revealing its in vivo pharmacological material basis. In addition, the application of RSBHXI strategy provides a new approach for metabolite identification of TCMPs and other complex mixture.

STUDY OF CHEMICAL FINGERPRINT FOR YUANHU ZHITONG TABLET BY UPLC/Q-TOF-MS

An ultra-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry (UPLC/Q-TOF-MS) was first employed to establish the chromatographic fingerprint and thereby identify the major components of Yuanhu Zhitong tablet (YZT), a traditional Chinese medicine. Fingerprint was established by analyzing 15 different batches of YZT, in which we obtained 17 common peaks with 18 constituents. Fifteen components were identified in the fingerprint with reference to the standards and literature data, including protopine, α-allocryptopine, coptisine, tetrahydropalmatine, tetrahydroberberine, corydaline, palmatine, berberine, byakangelicol, dehydrocorydaline, oxypeucedanin, imperatorin, phelloptorin, isoimperatorin, and osthole. Each of these was separated by using a Waters Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm) column with linear gradient elution of 0.2% formic acid water-acetonitrile. Meanwhile, the hierarchical cluster analysis was applied to evaluate the variation of the YZTs based on the characteristics of the common peaks in the UPLC/Q-TOF-MS profiles. This established method with high sensitivity and high speed may become a powerful tool for quality evaluation of YZT and related preparation.