Characterization of the multiple chemical components of Glechomae Herba using ultra high performance liquid chromatography coupled to quadrupole‐time‐of‐flight tandem mass spectrometry with diagnostic ion filtering strategy

Glechomae Herba is a traditional Chinese medicine used for the treatment of urolithiasis, cholelithiasis, and urinary tract infections in China. Identification of chemical constituents is helpful to discover the potential active ingredients. However, this significant work is stymied by complex chemical constituents. Therefore, an ultra high performance liquid chromatography coupled to quadrupole‐time‐of‐flight tandem mass spectrometry analysis with diagnostic product ions and neutral loss filtering strategy was established for chemical profiling of Glechomae Herba. The diagnostic product ions and neutral loss filtering strategy simplified spectral elucidation. A total of 120 compounds, including 10 chlorogenic acids, 10 gallic acids, 21 phenylpropionic acids, and 77 flavonoids, were reasonably identified in Glechomae Herba. Sixty‐five constituents were first discovered in Glechomae Herba. Four types of chlorogenic acids (caffeoylquinic acid, feruloylquinic acid, p‐coumaroylquinic acid, and di‐caffeoylquinic acid), three types of galloylglucoses (di‐O‐galloyl‐glucose, tri‐O‐galloyl‐glucose, and tetra‐O‐galloyl‐glucose), three types of phenylpropionic acid skeletons (p‐coumaric acid, caffeic acid, and rosmarinic acid) and five types of flavonoid aglycone skeletons (apigenin, kaempferol, luteolin, quercetin, and chrysin) were identified in Glechomae Herba. The results indicated that the developed strategy was feasible and rational technique for identifying the complex chemical constituents in Glechomae Herba.     

A novel strategy for target profiling analysis of bioactive phenylethanoid glycosides in Plantago medicinal plants using ultra-performance liquid chromatography coupled with tandem quadrupole mass spectrometry

Phenylethanoid glycosides are a group of phenolic compounds with diverse biological activities such as hypotensive, diuretic, and hypoglycemic effects. In this study, a target profiling analysis approach using ultra-performance liquid chromatography coupled with tandem quadrupole mass spectrometry (MS) was established on the basis of parent ion scanning for m/z 161, the characteristic product ion for phenylethanoid glycosides. It was successfully employed to discriminate the chemical composition of phenylethanoid glycosides between Plantaginis Herba and Plantaginis Semen, two medicinal parts of Plantago plants, which are widely used as herbal medicine in China. Totally, 34 phenylethanoid glycosides were characterized and tentatively identified by their retention times, MS, and tandem quadrupole MS (MS/MS) data. Combined with chemometrics analysis of principal component analysis and orthogonal projection to latent structural discriminate analysis, eight of them, especially acteoside and plantamajoside, were picked out and contributed to the chemical distinction between Plantaginis Herba and Plantaginis Semen, which might be responsible for the differences in diuretic and hypotensive effects between the two medicinal parts. This new approach for target profiling provides not only a novel idea for specific analysis of active chemical constituents in the same type, but also a promising and reference method for quality evaluation of traditional Chinese medicines.     

Application of characteristic fragment filtering with ultra high performance liquid chromatography coupled with high‐resolution mass spectrometry for comprehensive identification of components in Schisandrae chinensis Fructus

An integrated strategy of characteristic fragment filtering combined with target database screening based on ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry was proposed for comprehensive profiling of components in Schisandrae chinensis Fructus. The strategy consisted of following five steps: (1) Representative standards were analyzed by ultra high performance liquid chromatography coupled with linear ion trap‐Orbitrap mass spectrometer for characteristic fragments and fragmentation rules of each structure type. (2) The raw data of 70% methanol extract was collected by ultra high performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry. (3) The chemical components database that consisted of names, chemical formulas and structures of potential components in Schisandrae chinensis Fructus was established by summarizing previous literature to screen the collected liquid chromatography with mass spectrometry data and obtain matched compounds. (4) Characteristic fragments, literature, and reference standards were used to verify the matches. (5) Characteristic fragment filtering combined with online database querying was used to deduce potential new compounds. As a result, a total of 94 compounds were identified or characterized and 16 of them were potential new compounds. The study provided a reference for comprehensive characterization of ingredients in herbal medicine and formed the foundation for pharmacodynamic study of Schisandrae chinensis Fructus.     

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.     

Qualitative and quantitative analysis of chemical constituents in Ardisiae Japonicae Herba

Ardisiae Japonicae Herba is a well‐known traditional Chinese medicine for the treatment of bronchitis conjunctivitis, pneumonia, and trauma. In this work, a high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was first established for the separation and structural identification of the chemical constituents in Ardisiae Japonicae Herba. A total of 15 compounds including coumarins, flavonoid glycosides, and catechins were identified or tentatively characterized based on their chromatographic behaviors and mass spectral fragmentation and by comparisons with the reference standards. Furthermore, a simple high‐performance liquid chromatography with diode array detection method was developed for the simultaneous determination of five major constituents. Results obtained from method validation, including linearity, precision, repeatability, stability, and recovery, showed that the established method was reliable and accurate. Bergenin and quercitrin were found to be the most abundant constituents and could be served as chemical markers for quality control of Ardisiae Japonicae Herba.     

Fragmentation patterns study of iridoid glycosides in Fructus Gardeniae by HPLC‐Q/TOF‐MS/MS

Iridoid glycosides (IGs), the major constituents in Fructus Gardeniae, have demonstrated various pharmacological activities, but there is no systematic chemical profile of IGs in Fructus Gardeniae in the published literature until now. Therefore, it is imperative that a rapid and sensitive high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐Q/TOF‐MS/MS) method is established for comprehensive characterization of IGs in Fructus Gardeniae. Firstly, the fragmentation patterns of six known IGs were investigated and proposed and further concluded the diagnostic fragment ions and characteristic fragmentation pathways. Then, based on the summarized fragmentation patterns and the known compounds in the literatures, the other IGs in Fructus Gardeniae were identified successively. As a result, a total of 20 IGs were identified, of which three pairs of epimers were structurally characterized and differentiated. More importantly, one compound, the isoshanzhiside methyl ester, was tentatively identified as a new compound. The results of this study demonstrate the superiority of HPLC‐MS with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of the multiple groups of constituents in Fructus Gardeniae. Copyright © 2014 John Wiley & Sons, Ltd.     

The profiling and identification of chemical components,prototypes and metabolites of Run‐zao‐zhi‐yang capsule in rat plasma,urine and bile by an UPLC‐Q‐TOF/MSE‐based high‐throughput strategy

Run‐zao‐zhi‐yang (RZZY) capsule, a traditional Chinese medicine formula, is popularly used for the treatment of dermatitis and eczema. However, few studies have been carried out on RZZY and its metabolites. In this study, we developed a three‐step strategy to rapidly characterize the chemical constituents and metabolites of RZZY using ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. A total of 41 chemical components were characterized from RZZY. Among these, there are 11 flavonoids, six alkaloids, six stilbene glycosides, five anthraquinones and 13 other compounds. In addition, 18 prototypes and 35 metabolites were detected in rat plasma, urine and bile. This study offers an applicable approach for high‐throughput profiling and identification of chemical components and metabolites derived from traditional Chinese medicine formula in vivo, and also provides essential data for exploring bioactive ingredients and action mechanisms of RZZY.     

超高效液相色谱-高分辨质谱联用结合整合过滤策略全面分析茶树花中化学成分

茶树花与茶鲜叶同为茶树的生物产出,但茶树花往往被视为茶叶生产过程中的废物被舍弃,造成了茶树花资源的极大浪费.目前对于茶树花中化学成分的分析主要集中在氨基酸、茶多酚等单一类型化学成分上,对于茶树花中多类化学成分的同时分析仍鲜见报道.研究者对于茶树花中所含化学成分的种类和含量不完全清楚,成为制约茶树花深度开发与利用的重要原...     

A target and nontarget strategy for identification or characterization of the chemical ingredients in Chinese herb preparation Shuang‐Huang‐Lian oral liquid by ultra‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry

A target and nontarget strategy based on in‐house chemical components library was developed for rapid and comprehensive analysis of complicated components from traditional Chinese medicine preparation Shuang‐Huang‐Lian oral liquid. The sample was analyzed by ultra‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry using generic acquisition parameters. Automated detection and data filtering were performed on the UNIFI™ software and the detected peaks were evaluated against an in‐house library. As a result, a total of 170 chemical components (110 target compounds and 60 nontarget ones) were identified or tentatively characterized, including 54 flavonoids, 30 phenylethanoid glycosides, 16 iridoid glycosides, 14 lignans, 32 organic acids, 19 triterpenoid saponins and five other types of compounds. Among them, 44 compounds were further confirmed by comparison with reference standards. It was demonstrated that this systematical approach could be successfully applied for rapid identification of multiple compounds in traditional Chinese medicine and its preparations. Furthermore, this work established the foundation for the further investigation on the metabolic fates of multiple ingredients in Shuang‐Huang‐Lian oral liquid.     

Chemical profiling of constituents of Smilacis glabrae using ultra-high pressure liquid chromatography coupled with LTQ Orbitrap mass spectrometry

A reliable LC-MS method has been applied for the separation and identification of major constituents of the rhizome of Smilacis glabrae. Identification of the constituents was carried out by interpretation of their retention time, and MS and MS/MS data, especially by comparing these with Sarcandra glabra under the same LC-MS conditions, as well as the data provided by the literature. Thirty-three compounds, including catechin derivatives, flavanonols, phenolic acid derivatives and phenylpropanoid glycosides were either identified or tentatively characterized. Among them, compound 12 was deduced to be a new phenylpropanoid-substituted catechin. Fragmentation behaviors of the three major categories of compounds were also investigated. This UPLC-PDA/ESI-MS(n) method was effective for the separation and identification of the constituents and could be the basis for the comprehensive quality control of Smilacis glabrae.     

On-line identification of sugarcane (Saccharum officinarum L.) methoxyflavones by liquid chromatography-UV detection using post-column derivatization and liquid chromatography-mass spectrometry

Sugarcane (Saccharum officinarum L., Gramineae) bagasse and leaves were investigated for their flavonoid content and transgenic sugarcane ("Bowman-Birk" and "Kunitz") was compared with non-modified ("control") plants. Analyses were carried out by high-performance liquid chromatography coupled to diode array UV detection (LC/UV), also using post-column addition of shift reagents, and tandem MS (atmospheric pressure chemical ionization-MS/MS and collision-induced dissociation-MS). On-line UV and MS data demonstrated the presence of methoxyflavone glycosides and aglycones in a total of seven compounds. Three naturally occurring flavones glycosides and two unusual erythro- and threo-diastereoisomeric flavolignan 7-O-glucosides were identified together with their aglycones.     

Chemical profiling and characterization of phenolic acids,flavonoids, terpene glycosides from Vangueria agrestis using ultra-high-performance liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry and metabolomics approach

Vangueria agrestis is a shrub indigenous to tropical Africa, belonging to family Rubiaceae and is traditionally used as a decoction for treatment of fever, pain, and malaria. This study was undertaken to investigate the chemical constituents based on precursor exact mass and fragment ion information. The chemical profiling and structural characteristics of chemical constituents from methanolic extracts of dried aerial parts and roots of V. agrestis and dietary supplements were analyzed using ultra-high-performance liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry coupled with UNIFI platform and multivariate analysis in both negative and positive ion modes. A non-targeted ultra-high-performance liquid chromatography-mass spectrometry analysis was carried out to profile the chemical constituents of crude extracts of V. agrestis, and 73 compounds, including reference compounds, were identified. The fragments of flavonoids, monoterpene, and triterpene glycosides revealed the characteristic cleavage of glycosidic linkages, and the fragmentation pattern provided the identity of the sugars. This analytical method provides a quick method for quality assessment of dietary supplements. Finally, a chemometrics approach with multivariate statistical tools was used to visualize the differences between root and aerial parts of plant samples and to find the potential chemical markers that differentiate among these parts of V. agrestis samples and dietary supplements.     

Qualitative and quantitative studies on chemical constituents of Ling-gui-zhu-gan decoction: In vitro and in vivo

Ling-gui-zhu-gan decoction has significant therapeutic effects in the treatment of diseases related to phlegm and fluid retention. In this study, we aimed to qualitatively characterize the chemical constituents of Ling-gui-zhu-gan decoction in vitro and in vivo by HPLC coupled to Fourier transform ion cyclotron resonance MS, and quantitively determine the contents of typical chemical constituents by HPLC method. As a result, a total of 75 chemical constituents were discovered including 37 flavonoids and their glycosides, 20 saponins, 9 sterols, 3 organic acids and their derivatives, 3 lactones, 2 coumarins, and 1 alcohol. Among them, 17 chemical constituents were specifically identified. Subsequently, an HPLC method was established for simultaneous determination of seven chemical constituents. Finally, a total of 40 prototype components were initially detected by HPLC-MS method in the biological samples of rats after their water extract was orally administrated. Among them, 29, 27, 12, and 32 prototype components were detected in plasma, bile, urine, and feces, respectively. Moreover, 34 metabolites, including 16 phase II metabolites, were detected for the first time. In conclusion, this study lays the foundation for the identification of chemical components in vitro and in vivo and the elucidation of the potential pharmacodynamic components of Ling-gui-zhu-gan decoction.     

Qualitative and quantitative analysis of chemical constituents in traditional Chinese medicinal formula Tong-Xie-Yao-Fang by high-performance liquid chromatography/diode array detection/electrospray ionization tandem mass spectrometry

Tong-Xie-Yao-Fang (TXYF), a famous traditional Chinese medicine formula, has efficient effects on treatment of the diarrhea-predominant irritable bowel syndrome (D-IBS), a disease with high incidence worldwide. However, the active principles for this complex formula have not been fully explored so far. In this paper, high-performance liquid chromatography coupled with diode array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) was applied for the qualitative and quantitative analysis of major chemical constituents in TXYF. Two monoterpene glycosides, one chromone and five polymethoxylated flavones were tentatively characterized based on the retention times, UV spectra and MS data. Fifteen compounds were unambiguously identified by comparison with reference standards. Constituents such as lactone and steroidal, which could not be found by single HPLC method due to the low content in the formula, were identified in this paper. Seven compounds (gallic acid, prim-O-β-D-glucosylcimifugin, paeoniflorin, cimifugin, naringin, hesperidin and 4'-O-β-D-glucosyl-5-O-methylvisamminol) were quantified by HPLC-DAD using a C18 column and gradient elution with acetonitrile and water-0.1% formic acid. The method exhibited intra- and inter-day precision of less than 2.35% and 3.14%, respectively. The LODs and the LOQs for the analytes were less than 0.47 and 1.82 μg ml(-1), respectively. The overall recoveries ranged from 96.82% to 102.47%, with the R.S.D. ranging from 1.17% to 3.94%. These results demonstrated that our present method was effective and reliable for comprehensive quality evaluation of TXYF. Meanwhile, the study might provide the chemical evidence for revealing the material basis of its therapeutic effects.     

Quantitative analysis of Cistanches Herba using high‐performance liquid chromatography coupled with diode array detection and high‐resolution mass spectrometry combined with chemometric methods

We aim to determine the chemical constituents of three species of Cistanches Herba using HPLC coupled with diode array detection and high‐resolution MS. Ten phenylethanoid glycosides were identified and further quantified as marker substances by HPLC coupled with diode array detection method. The separation was conducted using an Agilent TC‐C18 column with 0.1% formic acid and methanol as the mobile phases under gradient elution. The analytical method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery, and subsequently applied to evaluate the quality of 36 batches of Cistanche plants. The chemometric procedures (i.e., hierarchical clustering analysis and principal component analysis) were used to compare different species of Cistanches Herba, leading to successful classification of the Cistanche samples in accordance with their origins. In conclusion, this study provides a chemical basis for quality control of Cistanches Herba.     

Identification of absorbed constituents and in vivo metabolites in rats after oral administration of Physalis alkekengi var. franchetii by ultra‐high‐pressure liquid chromatography quadrupole time‐of‐flight mass spectrometry

The calyces of Physalis alkekengi var. franchetii (Chinese Lantern, JDL) are well‐known as traditional Chinese medicine owing to its various therapeutic effects. However, the bioactive constituents responsible for the pharmacological effects of JDL and their metabolites in vivo are still unclear to date. In this paper, an ultra‐high‐pressure liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (UHPLC/Q‐TOF‐MS/MS) method was established to identify absorbed constituents and in vivo metabolites in rat biological fluids after oral administration of JDL. Based on the proposed strategy, 33 compounds were observed in dosed rat biosamples. Twelve of 33 compounds were indicated as prototype components of JDL, and 21 compounds were predicted to be metabolites of JDL. Finally, the metabolic pathways were proposed, which were glucuronidation, sulfation, methylation and dehydroxylation for flavonoid constituents and sulfonation and hydroxylation for physalin consitituents. This is the first systematic study on the absorbed constituents and metabolic profiling of JDL and will provide a useful template for screening and characterizing the ingredients and metabolites of traditional Chinese medicine.     

Urinary exposure marker discovery for toxicants using ultra-high pressure liquid chromatography coupled with Orbitrap high resolution mass spectrometry and three untargeted metabolomics approaches

Human biomonitoring is the assessment of actual internal contamination of chemicals by measuring exposure markers, chemicals or their metabolites, in human urine, blood, serum, and other body fluids. However, the metabolism of chemicals within an organism is extremely complex. Therefore, the identification of metabolites is often difficult and laborious. Several untargeted metabolomics methods have been developed to perform objective searching/filtering of accurate-mass-based LC-MS data to facilitate metabolite identification. In this study, three metabolomics data processing approaches were used for chemical exposure marker discovery in urine with an LTQ-Orbitrap high-resolution mass spectrometry (HRMS) dataset; di-isononyl phthalate (DINP) was used as an example. The data processing techniques included the SMAIT, mass defect filtering (MDF), and XCMS Online. Sixteen, 83, and 139 probable DINP metabolite signals were obtained using the SMAIT, MDF, and XCMS procedures, respectively. Fourteen probable metabolite signals mined simultaneously by the three metabolomics approaches were confirmed as DINP metabolites by structural information provided by LC-MS/MS. Among them, 13 probable metabolite signals were validated as exposure-related markers in a rat model. Six (m/z 319.155, 361.127, 373.126, 389.157, 437.112 and 443.130) of the 13 exposure-related DINP metabolite signals have not previously been reported in the literature. Our data indicate that SMAIT provided an efficient method to discover effectively and systematically urinary exposure markers of toxicant. The DINP metabolism information can provide valuable information for further investigations of DINP toxicity, toxicokinetics, exposure assessment, and human health effects.     

Characterization of chemical constituents in Rhodiola Crenulate by high‐performance liquid chromatography coupled with Fourier‐transform ion cyclotron resonance mass spectrometer (HPLC‐FT‐ICR MS)

In this work, an approach using high‐performance liquid chromatography coupled with diode‐array detection and Fourier‐transform ion cyclotron resonance mass spectrometer (HPLC‐FT‐ICR MS) for the identification and profiling of chemical constituents in Rhodiola crenulata was developed for the first time. The chromatographic separation was achieved on an Inertsil ODS‐3 column (150 mm × 4.6 mm,3 µm) using a gradient elution program, and the detection was performed on a Bruker Solarix 7.0 T mass spectrometer equipped with electrospray ionization source in both positive and negative modes. Under the optimized conditions, a total of 48 chemical compounds, including 26 alcohols and their glycosides, 12 flavonoids and their glycosides, 5 flavanols and gallic acid derivatives, 4 organic acids and 1 cyanogenic glycoside were identified or tentatively characterized. The results indicated that the developed HPLC‐FT‐ICR MS method with ultra‐high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents in R. crenulata. And it provides a helpful chemical basis for further research on R. crenulata. Copyright © 2016 John Wiley & Sons, Ltd.