Identification of anti‐inflammatory active ingredients from Tumuxiang by ultra‐performance liquid chromatography/quadrupole time‐of‐flight‐MSE

The dried roots of Inula helenium L. (IH) and Inula racemosa Hook f. (IR) are used commonly as folk medicine as ‘tumuxiang’ (TMX). The mixing and sharing of IH and IR in clinical use is a universal phenomenon. Modern pharmacological studies confirmed that IH and IR display anti‐inflammatory activities. However, the difference in anti‐inflammatory pharmacodynamic substances between these two herbs is still unknown. In the present study, the fingerprints of 18 IH and nine IR samples were established using UPLC/QTOF‐MS^E. A dimethylbenzene‐induced mouse ear vasodilation model was applied in evaluating the anti‐inflammatory properties of all 27 samples. Then, the spectrum–efficacy model between chemical characteristic peaks and anti‐inflammatory activities was investigated using principal component regression and partial least squares. Finally, the combination of UNIFI Scientific Information System with a library search of traditional Chinese medicines was employed to automatically characterize the peaks. UNIFI identified a total of 80 chemical components. Among the components, the 53 characteristic peaks showed correlation with anti‐inflammatory activities, pointing to phenolic and organic acids as primary anti‐inflammatory ingredients of TMX. This approach can efficiently and intelligently facilitate the identification of bioactive components from traditional Chinese medicine.     

Qualitative analysis of chemical constituents in traditional Chinese medicine analogous formula cheng‐Qi decoctions by liquid chromatography–mass spectrometry

Cheng‐Qi decoctions (CQs), a group of analogous formulas, are well‐known traditional Chinese preparations used as purgative remedies to treat ‘internal heat’‐induced symptoms, which manifest as a bloated and painful abdomen, hard stools, fever and other clinical observations. In this study, HPLC‐ESI‐MS/MS and UPLC‐TOF‐MS were employed for separation and structural identification of constituents in CQs. As a result, a total of 90 compounds, including seven anthraquinones, 39 flavones, 21 glycosides, 11 stilbene glycosides, six organic acids, five coumarins and one lignans, were detected and tentatively identified in CQs extracts. The characterization results shed some light on the scientific foundation for clinical application of the CQ analogous formulas. Our results also indicate that the HPLC‐MS method is useful for the systemic identification of major constituents in traditional Chinese medicine formulas. Copyright © 2015 John Wiley & Sons, Ltd.     

Material basis studies of anti‐Influenza A active ingredients in Tanreqing Injection

Tanreqing Injection (TRQ) has been used primarily in treating infections of the upper respiratory tract and serious influenza in China, as a classical compound herbal recipe. TRQ had been demonstrated to have effects of clearing heat, eliminating phlegm, detoxification, reducing inflammation and alleviating cough. The survival rate, histopathology of lungs and viral titers in mice were evaluated in this study to verify the curative effect of TRQ. However, there is not enough information about the components. In the present study, a high‐performance and practical LC/QTOF/MS method was developed for characterization and identification of the natural ingredients in TRQ. A total of 60 compounds, including 10 amino acids, 10 iridoid glucosides, 14 flavonoids, 13 other phenolic compounds, 10 steroid acids and three other compounds, were characterized and identified. We also confirmed the material basis of anti‐Influenza A active ingredients in TRQ. Therefore, we have developed an accurate analytical method. LC/QTOF/MS could be applied for identification the complex components in traditional Chinese medicine.     

UPLC‐QTOF‐MSE‐based diagnostic product ion filtering to unveil unstable C6‐C2 glucoside conjugates in Forsythia suspensa

Forsythia suspensa contains C₆‐C₂ glucoside conjugates (CCGCs) that are chemically unstable, thereby hindering their isolation and purification. In the present study, ultra‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UPLC‐QTOF) was utilized to screen and identify unstable CCGCs in the fruits and leaves of F. suspensa without any tedious isolation and purified process based on independent information acquisition (also called MS^E) and individual MS/MS experiments. Diagnostic product ion filtering (DPIF) was further applied to mine unknown analogs in MS^E high energy levels based on characteristic m/z of key substructures. A modified nomenclature for CCGCs is hereby proposed to facilitate discussions. Possible fragmentation pathways of major types of known CCGCs were proposed and used for deducing their structures. A total of 8 potentially new CCGCs were discovered and initially identified. The accuracy of their identification was further verified by structural elucidation of 3 unstable CCGCs isolated from the fruits of F. suspensa using 1D and 2D‐NMR spectroscopy. The established UPLC‐QTOF‐MS^E‐based DPIF technique facilitates the rapid discovery and direct identification of unstable CCGCs in fruits and leaves of F. suspensa .     

Rapid identification and comparative analysis of the chemical constituents and metabolites of Phellodendri amurensis cortex and Zhibai dihuang pill by ultra‐performance liquid chromatography with quadrupole TOF‐MS

Zhibai dihuang pill is a famous traditional Chinese medicine formula containing the herbal medicine Phellodendri amurensis cortex. In this work, an integrative pattern recognition approach including principal component analysis and orthogonal partial least squared discriminant analysis based on ultra‐performance liquid chromatography coupled to MS was successfully applied for the rapid discovery of natural compounds from herbal medicines. In a 24 min analysis, 93 compounds were identified or tentatively characterized from Zhibai dihuang pill based on their fragmentation behaviors, 23 of which were from Phellodendri amurensis cortex. A total of 26 metabolites in plasma were identified from Phellodendri amurensis cortex in Zhibai dihuang pill formula, among them, 12 prototypes and 14 metabolites through metabolic pathways of demethylation, methylation, hydrolysis, sulfate conjugation, and glucuronide conjugation, which were seven metabolites more than that of the single drug, suggesting the importance of the compatibility of traditional Chinese medicine. The present study provided important structural information on the metabolism of Zhibai dihuang pill. Furthermore, the results of this work have demonstrated the feasibility of ultra‐performance liquid chromatography coupled to MS for the rapid and reliable characterization of metabolites from herbal medicines. Based on these results, this method could be a novel approach for identifying potentially bioactive components in other traditional Chinese medicines.     

Chemical composition differentiation of Shen‐Shuai‐Ning granule between combined decoction and separated decoction using HPLC‐DAD‐ESI‐QTOF‐MS

Shen‐Shuai‐Ning (SSN) granule, a traditional Chinese medicine formula, is widely used in clinical practice for treating chronic renal failure. However, its detailed chemical profile is unknown. Here, HPLC‐ESI‐QTOF‐MS was employed for the systematic chemical analysis of SSN. A total of 52 compounds were identified and the characteristic ions of the compounds were described. Furthermore, chemical consistency between the combined decoction and the separated decoction of SSN was evaluated using HPLC‐DAD. A chemical comparison between two preparations of SSN granule (combined decoction and separated decoction of Coptides Rhizoma) indicated a significant difference in the content of many compounds, including salvianolic acid A, salvianolic acid B, berberine, palmatine and epiberberine. As a result, separated decoction of Coptides Rhizoma would lead to a significantly decrease in depsides in Salviae Miltiorrhizae Radix et Rhizoma and an increase in alkaloids in Coptidis Rhizoma.     

Method for rapidly discovering active components in Yupingfeng granules by UPLC‐ESI‐Q‐TOF‐MS

Yupingfeng granules (YPFG) were isolated from a traditional Chinese medicine (TCM) formulation composed of three herbs (Astragali Radix, Atractylodis Macrocephalae Rhizoma, and Saposhnikoviae Radix). This formulation is used in TCM to tonify qi, and it can help strengthen exterior and reduce sweating. Nevertheless, the active components of YPFG remain unclear. In this study, the chemical constituents of YPFG were systematically characterized by ultra‐performance liquid chromatography coupled with electrospray ionization/ quadrupole time‐of‐flight mass spectrometry (UPLC‐ESI‐Q‐TOF‐MS). Fifty‐eight compounds, namely, 20 flavonoids, 19 saponins, nine organic acids, four volatile coumarins, three lactones, one alkaloid, and two other components, were identified. In addition, the constituents of YPFG with the potential for in vivo bioactivities following oral administration were investigated in Sprague–Dawley rats. Thirteen compounds, namely, 11 flavonoid‐related and 2 saponin‐related components, were detected in rat plasma. After enriching flavonoids and saponins in YPFG by extraction, the extracts and YPFG were administrated to immunosuppressed rats, respectively. Plasma samples were analyzed by UPLC‐ESI‐Q‐TOF‐MS, and principal component analysis (PCA) confirmed that the extracts had similar effects to YPFG. This method could discover active ingredients in YPFG quickly and provide a scientific basis for quality control and mechanism research.     

Qualitative and quantitative analysis of the major constituents in Acorus tatarinowii Schott by HPLC/ESI‐QTOF‐MS/MS

Acorus tatarinowii Schott (ATS) is a well‐known traditional Chinese medicine (TCM) for the treatment of epilepsy, amnesia and insomnia. In this study, a methodology utilizing high‐performance liquid chromatography (HPLC) coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐QTOF‐MS/MS) was established for the separation and structural identification of the major chemical constituents in ATS for the first time. Overall, 46 major constituents including flavonoid glycosides, phenylpropane derivatives, amides and lignans were identified or tentatively characterized. Seven major constituents, including four phenylpropane derivatives and three lignans, were further quantified as marker substances, which showed good linearity within the test ranges. These results indicated that the developed quantitative method was linear, sensitive, and precise for quality control of ATS. Copyright © 2014 John Wiley & Sons, Ltd.     

Plasma pharmacochemistry combined with microdialysis to screen potential bioactive components and their metabolites in Anemarrhena asphodeloides saponin extract using ultrahigh‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

Although various techniques have been employed to analyze drug metabolites, the metabolism of multicomponent herbal medicine has seldom been fully addressed. In contrast to chemical drugs, a number of compounds in herbal medicine could get into circulation and then be metabolized. Metabolism study on active constituents in herbal medicine is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of herbal medicine. The present work aims to elucidate the multicomponent metabolic characteristics of a herbal medicine by the combination of plasma pharmacochemistry and microdialysis sampling. Anemarrhena asphodeloides, a well‐known traditional Chinese medicine, was chosen as a model. After oral administration of A. asphodeloides saponin extract to rats, microdialysis samples were collected continuously in the jugular vein and analyzed by ultrahigh‐performance LC/quadrupole‐TOF MS. The identification of compounds in biosamples was achieved by accurate mass measurement and detailed fragmentation pathway analysis. The results showed that unbound constituents in blood circulation of the rat included seven parent saponins and six metabolites, which might be the potential active components in vivo. Among which, three metabolites have not been previously reported and were identified in this study. It is the first report on systemic metabolism of total saponins of A. asphodeloides in mammalian plasma.     

Identification of interactions between multiple components in Socheongryong‐tang using a plant profiling approach

Traditional herbal medicine consists of multiple components. There are interactions among the components, which affect both potency and toxicity. The preparation of herbal medicines can be a cause of interactions between multicomponents in herbs. To demonstrate the differences in multiherb interactions based on the preparation methods, the changes in the active components in the different preparations of Socheongryong‐tang (SCRT) were evaluated using metabolomics profiling. We performed multicomponent profiling of the decoction of SCRT (SCRTD) and individual herb mixture (SCRTM) using ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (UPLC–QTOF–MS). Active compounds from SCRTD and SCRTM were identified using multivariate analysis, and the activities between the two groups were compared. We also evaluated the anti‐inflammatory effect of SCRT through investigating the protein expression of iNOS and COX‐2 in lipopolysaccharide‐induced macrophage RAW 264.7 cells in both groups. From the multivariate analysis, 53 active compounds that have different intensities between SCRTD and SCRTM were identified. The intensities of those components, such as ephedrines, glycyrrhizic acid, 6‐gingerol and (2E,4E,8Z,10E)‐N‐isobutyl‐2,4,8,10‐dodecatetraenamide, which is newly identified in Asiasarum heterotropoides, were mostly higher in SCRTD than in SCRTM, which was related to the anti‐inflammatory effect. From the iNOS inhibition test, it was found that SCRTD had a stronger anti‐inflammatory effect than SCRTM. It was demonstrated that multicomponent interactions can be changed by the preparation method, and finally the anti‐inflammatory effect in SCRT can be affected.     

Bioactivity‐based ultra‐performance liquid chromatography‐coupled quadrupole time‐of‐flight mass spectrometry for NF‐κB inhibitors identification in Chinese Medicinal Preparation Bufei Granule

Traditional Chinese medicine (TCM) preparations have become effective treatments for many diseases. However, their active ingredients are still uncertain and difficult to identify. In this study, we propose a strategy that integrates ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) and bioactive (NF‐κB inhibitor) luciferase reporter assay systems for the rapid determination of various anti‐inflammatory compounds of TCM preparations. In this way, Bufei Granule (BFG), a TCM preparation used for the clinical therapy of asthma, was analyzed by the two ways of component identification and activity detection. Potential anti‐inflammatory constituents were screened by NF‐κB activity assay systems and simultaneously identified according to the mass spectrometry data. Three structural types of NF‐κB inhibitors (caffeic acid derivatives, flavonoids and Pentacyclic triterpenes) were characterized. Further cytokine detection confirmed the anti‐inflammatory effects of the potential NF‐κB inhibitors. Compared with conventional chromatographic separation and inhibitory activity detection, integrating UPLC/Q‐TOF‐MS identification and virtual validation was more convenient and more reliable. This strategy clearly demonstrates that MS data‐based fingerprinting is a meaningful tool not only in identifying constituents in complex matrix but also in directly screening for powerful trace ingredients in TCM preparations. Copyright © 2016 John Wiley & Sons, Ltd.     

超高效液相色谱-四极杆串联飞行时间质谱分析黄酮类化合物及小毛茛茎叶的成分

为了探索液相色谱-质谱联用(LC-MS)技术在快速识别中药及天然产物成分中的应用,以黄酮对照品为研究前体,药用植物小毛茛为研究对象,采用超高效液相色谱/二极管阵列检测器-电喷雾四极杆串联飞行时间质谱(UPLC/DAD-ESI/Q-TOF MS)分析了黄酮类化合物同系物及同分异构体的色谱、质谱特性。结果显示:黄酮氧苷和黄酮碳苷的紫外吸收光谱及二级质谱具有显著性差异,糖苷化位置同保留时间、二级质谱碎片及相对丰度具有相关性。将该方法应用于小毛茛茎叶醇提液的分析,结合其酸水解液的分析,解析了22个黄酮醇糖苷和3个苷元。方法简便,具有可操作性。     

Characterization of the chemical constituents in Da‐Huang‐Gan‐Cao‐Tang by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry and liquid chromatography coupled with ion trap mass spectrometry

In this work, the chemical constituents in Da‐Huang‐Gan‐Cao‐Tang, a traditional Chinese formula, were studied by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry and liquid chromatography coupled with ion trap mass spectrometry for the first time. Among the 146 compounds detected in Da‐Huang‐Gan‐Cao‐Tang, 104 compounds were identified unambiguously or tentatively based on their accurate molecular weight and multistage MS data, including one potential novel compound and two reported in Glycyrrhiza genus for the first time. The possible fragmentation pathways were proposed and fragmentation rules of the major types of compounds were concluded. This study provided an example to facilitate the tedious identification of chemical composition in traditional Chinese medicine, and maybe a promising reference approach to research the analogous formulae.     

Ultra‐performance liquid chromatography coupled with electrospray ionization/quadrupole time‐of‐flight mass spectrometry for the rapid analysis of constituents in the traditional Chinese medical formula Danggui San

In this work, ultra‐performance LC with ESI quadrupole TOF‐MS (UPLC–ESI‐Q‐TOF‐MS) and automated MetaboLynx analysis was used to rapidly separate and identify the chemical constituents of Danggui San, a traditional Chinese medical formula. The analysis was performed on a Waters UPLC BEH C₁₈ column using a gradient elution system. A hyphenated ESI and Q‐TOF analyzer was used for the determination of the accurate mass of the protonated or deprotonated molecule and fragment ions in both positive and negative modes. Based on retention times, accurate mass, and the mass spectrometric fragmentation characteristics, a total of 47 compounds distributed over the chemical groups of phthalides, flavonoids, monoterpene glycosides, sesquiterpenoids, phenolics, and alkaloids, were simultaneously separated within 18 min and identified or tentatively elucidated in Danggui San for the first time. UPLC–ESI‐Q‐TOF‐MS analysis revealed the complexity of the chemical composition of this formula. The method developed is rapid, accurate, reliable, and highly sensitive to characterize the chemical constituents of Danggui San.     

Characterization of protostane triterpenoids in Alisma orientalis by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

A reliable and sensitive ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) method has been optimized and established for analysis of protostane triterpenoids in a commonly used traditional Chinese herbal medicine Alisma orientalis (Sam.) Juzep. The separation of crude extract of A. orientalis was achieved on a Waters ACQUITY HSS T3 column (100 mm × 2.1 mm, 1.8 µm) eluting with 0.1% (v/v) formic acid/acetonitrile. A total of 20 protostane triterpenoids including 19 known compounds and a new one were well separated within 7 min. The collision‐induced dissociation (CID) tandem mass spectrometric (MS/MS) fragmentation patterns of protostane triterpenoids was firstly reported in this study. The hydrogen rearrangement at the C‐23‐OH leads to dissociation of the bond between C‐23 and C‐24 in the protostane triterpenoid skeleton during the CID process. This dissociation was the characteristic CID fragmentation pathway of this class of triterpenoids, and was useful for further differentiation of some positional isomers which contain an acetyl unit on the C‐23 or C‐24 position. The identities of isolated compounds were identified by comparing their retention times and CID fragmentation behaviors with those of reference standards or tentatively assigned by matching the empirical molecular formulae with those reported in the literature. It is concluded that this newly established UPLC/Q‐TOF‐MS method is a powerful approach for structural elucidation of protostane triterpenoids isolated from A. orientalis. Copyright © 2010 John Wiley & Sons, Ltd.