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Monitoring the ingredient change during the production of Tan Re Qing capsules from Scutellariae Radix by HPLC-MS/MS

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Abstract

Tan Re Qing (TRQ), a traditional Chinese medicine formula, is composed of Scutellariae Radix and other four herbs (bear bile powder, Caprae Hircus Cornu, Lonicerae japonicae Flos and Forsythiae Fructus) used for the treatment of bronchitis and upper respiratory infection. Chemical constituents in the herb always changed in content during processing. So, in this study, the ingredient change was monitored from three froms of Scutellariae Radix, including original herb, intermediate and product (TRQ capsules) with the same batch by HPLC MS/MS. A total of 98 compounds, including 69 flavonoids, 5 phenylethanoid glucosides, 6 other compounds and 18 unknown components, were tentatively identified in Scutellariae Radix. Most minor components in Scutellariae Radix were disappeared during preparation process, and only 17 of these compounds were detected in TRQ capsules. 5,2’-Dihydroxy-7,8-dimethoxyflavone and one unknown compound were detected in the original herb and intermediate, but disappeared in the TRQ capsules. These findings indicated the productive process control is vital for risk assessment and chemical material basis, as well as the basis for quality control of TRQ capsules.

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Much Literature is available with respect to the collision-induced dissociation (CID) of different subtypes of flavonoid aglycones. However, a simultaneous comparison study of the common flavonoid subtypes under unified conditions is rarely reported, which is of crucial significance for their rapid structural elucidation. This study depicted the negative CID of 40 free flavonoids (comprising nine different subtypes) utilizing electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MSn). High-accuracy quadrupole time-of-flight mass spectrometry was employed to confirm the chemical formula of proposed key fragments. Specific fragmentation features were discovered among different flavonoid subtypes in terms of the essential collision energy, base peak, RDA fragmentation, and diversity of fragmentation pathways: CID of flavanones, chalcones and flavanonols was in need of lower Levels of collision energy [<40%) and exhibited simplified fragmentation pathways; overriding neutral loss of CO2 was detected for flavones, aurones and xanthones, while isoflavones preferred neutral loss of CO; 1,2A- and 1,3A- fragments were respectively dominant for flavonols and flavanones/chalcones, white homoisoflavones underwent specific radical elimination of ring B or methylene-ring B. These differentiated fragmentation characteristics integrating UV absorption resulted in the establishment of a decision tree to characterize and distinguish the common flavonoid subtypes. This study firstly revealed remarkable differentiations in the negative CID behavior of nine common flavonoid aglycone subtypes and the proposed determination strategy would much benefit the screening and rapid characterization of flavonoids from complicated matrixes by tandem mass spectrometry.
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Gegen-Qinlian-Wan (GQW) is a popular traditional Chinese patent medicine for the treatment of diarrhea. It is composed of four herbal medicines, Puerariae Radix, Scutellariae Radix, Coptidis Rhizoma, and Glycyrrhizae Radix. In this study, a rapid and sensitive method based on ultra high-performance liquid chromatography coupled with diode-array detection and quadrupole time-of-flight mass spectrometry (UHPLC-DAD-qTOF-MS) was established to characterize the chemical constituents and rats metabolites of GQW. Samples were separated on an Agilent Zorbax Eclipse Plus-C(18) column (100mm×2.1mm, 1.8μm) by gradient elution using acetonitrile and water containing 0.1% formic acid as the mobile phase. On the basis of UV and qTOF high-accuracy mass spectral analysis, a total of 62 compounds were identified or tentatively characterized from GQW, including 42 flavonoids, 8 alkaloids, 6 triterpenoids, 3 phenylethanoid glycosides, and 3 other types. Among them, 27 compounds were confirmed by comparing with reference standards. Furthermore, metabolites in rats plasma and urine after oral administration of GQW were also analyzed. A total of 42 compounds were identified, including 29 prototypes and 13 metabolites through metabolic pathways of demethylation, methylation, hydrolysis, sulfate conjugation, and glucuronide conjugation. Glucuronidated flavonoids were the main constituents in the plasma, and were then transformed into aglycones and excreted from urine. This is the first systematic study on the chemical constituents and metabolic profiling of GQW.
Article
A high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS2) method was firstly developed for a chemical fingerprint analysis of Scutellariae Radix and rapid identification of major compounds in the fingerprints. The experimental data for chromatography were used for hierarchical clustering analysis and similarity calculation, and those for UV and MS spectra were applied for identifying characteristic peaks. Twenty samples including S. baicalensis Georgi., S. viscidula Bge. and S. amoena C. H. Wright were classified into three groups. By comparing the UV and MS spectra data with those of the authentic standards and literature, 20 main peaks in the fingerprints were identified for the first time. The developed fingerprint assay was specific and could be readily utilized for comprehensive evaluation of Scutellariae Radix.
Article
Eleven naturally occurring flavonoid aglycones, belonging to the representative flavone, flavonol, and flavanone types were separated by high performance liquid chromatography and analyzed on-line with negative ion electrospray ionization tandem mass spectrometry (ESI-MS/MS). In order to resolve the MS/MS spectra obtained, each compound was reinvestigated by direct loop injections using an ion trap mass spectrometer. The MSn spectra obtained allowed us to propose plausible schemes for their fragmentation supported by the analysis of five complementary synthetic flavonoid aglycones. The negative ion ESI-MS/MS behavior of the different aglycones investigated in this study revealed interesting differences when compared with the previously described patterns obtained using various ionization techniques in positive ion. Thus, concerning the retro Diels-Alder (RDA) fragmentation pathways, several structurally informative anions appeared highly specific of the negative ion mode. In addition, a new lactone-type structure, instead of a ketene, was proposed for a classic RDA diagnostic ion. We also observed unusual CO, CO2, and C3O2 losses which appear to be characteristic of the negative ion mode. All these results and these unusual neutral losses show that the negative ion mode was a powerful complementary tool of the positive ion mode for the structural characterization of flavonoid aglycones by ESI-MS/MS.
Article
Radix Scutellariae is the dried root of the medicinal plant Scutellariae baicalensis Georgi. It exhibits a variety of therapeutic effects and has a long history of application in traditional formulations as well as in modern herbal medications. It has been confirmed that flavonoids are the most abundant constituents and induce these therapeutic effects. Six flavones are proven to be the major bioactive flavones in Radix Scutellariae existing in the forms of aglycones (baicalein, wogonin, oroxylin A) and glycosides (baicalin, wogonoside, oroxylin A-7-glucuronide). All six flavones are pharmacologically active and show great potential in the treatment of inflammation, cancers and virus-related diseases. The current review covers the preparation of the herb Radix Scutellariae, quantification of its major bioactive ingredients, and pharmacological effects of the proposed six bioactive flavones. In addition, this review summarizes the pharmacokinetic profiles of the bioactive flavones reported so far that could be used for further improvement of their pharmacokinetic study. Moreover, due to abundant co-occurring bioactive components in Radix Scutellariae, our review further documents the pharmacokinetic interactions among them.
Article
Scutellaria (HUANG QIN) (Lamiaceae), which includes about 350 species commonly known as skullcaps, is widespread in Europe, the United States and East Asia. Some species are taken to clear away the heat-evil and expel superficial evils in traditional Chinese medicine (TCM). The present paper reviews the ethnopharmacology, the biological activities and the correlated chemical compounds of Scutellaria species. More than 295 compounds have been isolated, among them flavonoids and diterpenes. Studies show that Scutellaria and its active principles possess wide pharmacological actions, such as antitumor, anti-angiogenesis, hepatoprotective, antioxidant, anticonvulsant, antibacterial and antiviral activities. Currently, effective monomeric compounds or active parts have been screened for pharmacological activity from Scutellaria in vivo and in vitro. Increasing data supports application and exploitation for new drug development.
Article
Scutellaria baicalensis Georgi is a well-known medicinal plant widely used in China and other East Asian countries. High performance liquid chromatography combined with diode array detection and electrospray ion trap mass spectrometry was used to determine the flavonoid profile of S. baicalensis. Under the optimized experiment conditions, 32 flavonoids were clearly detected. Eighteen main ones were doubtless identified by comparing their retention time, UV and MS (MS(n)) data with isolated or commercial standards. The UV characteristics of these 18 known standards were studied in detail. The rules summarized provided valuable indications for the subsequent on-line identification processes. By interpreting both the MS and the UV data in detail, other 13 minor flavonoids in S. baicalensis were on-line identified successfully.
Article
High-performance liquid chromatography coupled to positive ion electrospray ionization tandem mass spectrometry (MS) and diode array detection was employed to identify the polyphenol C-glycosides in the extract of Swertia franchetiana, a traditional Chinese/Tibetan herb. The neutral loss scan of the extract of S. franchetiana using the characteristic losses of 120 and 150 u provided a detailed profile of the polyphenol C-glycosides in the complex mixture. On-line UV spectroscopy along with MS-MS and MS-MS-MS mass spectra analysis produced with and without in-source collision induced dissociation was contributed to discriminate and identify the polyphenol C-glycosides. Three xanthone C-glycosides (i.e., mangiferin, isomangiferin, and 1,6,7-trihydroxyl-2-C-glucosexanthone) and three flavone C-glycosides (i.e., isoorientin, isovitexin, and swertisin) were tentatively identified. Isomangiferin and 1,6,7-trihydroxyl-2-C-glucosexanthone were for the first time found in this plant.
Article
Fragmentation pathways of nine flavone compounds have been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn). Analyzing the product ion spectra of flavonoids and aglycones, we observed some diagnostic neutral losses, such as CH3, H2O, residue of glucose and gluconic acid, which are very useful for the identification of the functional groups in the structures. Furthermore, specific retro Diels-Alder (RDA) fragments for flavones with different hydroxyl substitution have also been discussed. The information is helpful for the rapid identification of the location site of hydroxyl substitution on flavones. Fragmentation pathways of C-glycosidic flavonoid have also been discussed using ESI-MSn, demonstrating ions [M-H-60]-, [M-H-90]-, [M-H-120]- are characteristic ions of C-glycosidic flavonoid. According to the fragmentation mechanism of mass spectrometry and HPLC-MS data, the structures of seven flavones in Scutellaria baicalensis Georgi have been identified on-line without time-consuming isolation. The HPLC-ESI-MSn method for analyzing constituents in the Scutellaria baicalensis Georgi has been established.
Article
The root of Scutellaria baicalensis, called Huangqin in Chinese, is one of the most commonly used traditional Chinese medicines for the treatment of hepatitis, tumors, diarrhea, and inflammatory diseases. The major chemical constituents of Huangqin are flavonoids. In the present paper, HPLC-DAD-ESI-MS(n) was used to analyze flavonoids in the roots of S. baicalensis. A total of 26 flavonoids were identified or tentatively characterized, including 5 C-glycosides, 12 O-glycosides, and 9 free aglycones. Two C-glycosides, apigenin-6-C-glucyl-8-C-arabinoside and chrysin-6,8-di-C-glucoside, together with some O-glycosides, are reported from S. baicalensis for the first time. This method is simple, reliable and sensitive, and could be used for the quality control of Huangqin and its related preparations.
Article
PHY906 is a Chinese medicine formulation prepared from four medicinal herbs for adjuvant cancer chemotherapy. In this paper, liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOFMS) was used to clarify the chemical composition of PHY906. The aqueous extract of PHY906 was separated on a Waters Atlantis C(18) column, and was eluted with acetonitrile/0.05% (v/v) formic acid. The separated compounds were identified with pure standards, or tentatively characterized by analyzing their mass spectra recorded in both negative and positive ion polarity modes. Further structural information was obtained from in-source fragmentation. Based on the LC/MS analysis, we proposed the structures for 64 bioactive compounds, including flavonoids, triterpene saponins, and monoterpene glycosides. All the compounds identified from PHY906 were further assigned in the four individual herbs, and some of them are reported for the first time.
Article
Six flavone mono-C-glucosides, four standards (beta-D-glucopyranosyl-(1 --> C-6)- and -(1 --> C-8)- apigenin and luteolin) and two others from lemon juice (beta-D-glucopyranosyl-(1 --> C-6)- and -(1 --> C-8)-diosmetin) have been studied in order to analyze their fragmentation patterns. Initial separation was carried out using high-performance liquid chromatography with diode-array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer. Several systematic differences between collision-induced dissociation tandem mass (CID-MS/MS) spectra of C-6- and C-8-isomers have been found and some general guidelines and two new diagnostic product ions have been proposed for the differentiation of C-6- and C-8-flavonoid glycosides. These results have been successfully applied to the characterization of two flavone C-glycosides found in lemon juice, and mass spectra of a flavone di-C-glycoside detected in lemon juice have been studied and interpreted.
Comparison of Chemical Compositions between Tanreqing Capsule and Tanreqing Injection by HPLC-MS/MS
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  • P Zhong
  • Y Xu
Preparation of Flavonoid Reference Standards from Scutellariae Radix under the Guidance of High Performance Liquid Chromatography-Mass Spectrometry Analysis. Chin
  • H Guo
  • X Yang
  • J Liu
  • W Zheng
Electrospray Ionization Mass Spectrometry for Analyses of Alkaloids in Sophoraflavescens ait
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  • F Han
  • P Du
  • Y Chen
Assay of Amino Acid in Cornu Caprae Hircus Extract
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  • D Xue
  • J Li
  • B Wang
  • Z Zhang
  • M Miao