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.     

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.     

Chemical profiling approach to evaluate the influence of traditional and simplified decoction methods on the holistic quality of Da‐Huang‐Xiao‐Shi decoction using high‐performance liquid chromatography coupled with diode‐array detection and time‐of‐flight mass spectrometry

Da‐Huang‐Xiao‐Shi decoction, consisting of Rheum officinale Baill, Mirabilitum, Phellodendron amurense Rupr. and Gardenia jasminoides Ellis, is a traditional Chinese medicine used for the treatment of jaundice. As described in “Jin Kui Yao Lue”, a traditional multistep decoction of Da‐Huang‐Xiao‐Shi decoction was required while simplified one‐step decoction was used in recent repsorts. To investigate the chemical difference between the decoctions obtained by the traditional and simplified preparations, a sensitive and reliable approach of high‐performance liquid chromatography coupled with diode‐array detection and electrospray ionization time‐of‐flight mass spectrometry was established. As a result, a total of 105 compounds were detected and identified. Analysis of the chromatogram profiles of the two decoctions showed that many compounds in the decoction of simplified preparation had changed obviously compared with those in traditional preparation. The changes of constituents would be bound to cause the differences in the therapeutic effects of the two decoctions. The present study demonstrated that certain preparation methods significantly affect the holistic quality of traditional Chinese medicines and the use of a suitable preparation method is crucial for these medicines to produce special clinical curative effect. This research results elucidated the scientific basis of traditional preparation methods in Chinese medicines.     

Chemical profiling and quantification of multiple components in Jin-Gu-Lian capsule using a multivariate data processing approach based on UHPLC-Orbitrap Exploris 240 MS and UHPLC-MS/MS

The Jin-Gu-Lian capsule, a Chinese Miao herbal compound, is widely used to treat rheumatoid arthritis. In this study, a rapid, selective, and sensitive UHPLC-Orbitrap Exploris 240 MS method was developed to analyze the chemical composition of Jin-Gu-Lian capsules. A total of 88 compounds were identified, including 23 flavonoids, 23 organic acids, 14 phenylpropanoids, 12 phenols, eight alkaloids, four terpenes, three quinones, and one ketone. Among these, 21 compounds were clearly detected based on a comparison with reference standards and selected as quality control markers. Thereafter, these compounds were simultaneously determined in the Jin-Gu-Lian capsules. The established method was successfully validated and applied for the simultaneous determination of 21 biologically active compounds in Jin-Gu-Lian capsules of 27 sample batches. Quantitative data of the analytes were analyzed using multivariate statistical analysis to determine the quality of the Jin-Gu-Lian capsules. Four compounds (JGLC6 [salidroside], JGLC8 [chlorogenic acid], JGLC12 [liriodendrin], JGLC19 [quercetin]) were identified as chemical markers for quality control of Jin-Gu-Lian capsules. Altogether, the established method was validated as a novel and efficient tool, that can be used for rapid analysis of Jin-Gu-Lian capsules. Accordingly, this study serves as a reference for scientific research on traditional Chinese and ethnic medicine.     

Rapid identification of chemical compositions in callicarpa kwangtungensis Chun by ultra‐high‐performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high‐resolution accurate mass spectrometry

Callicarpa kwangtungensis Chun is a traditional Chinese medicine that has various therapeutic effects. Despite its wide use in Chinese medicine, the study is still quite limited, especially its chemical compositions. In this research, an ultra‐high‐pressure liquid chromatography coupled with Q Exactive hybrid quadrupole‐orbitrap high‐resolution accurate mass spectrometry tandem mass spectrometry method was utilized to analyze its chemical compositions for the first time. As a result, a total of 124 compounds, including 20 phenylethanoid glycosides, 31 flavonoids, 36 organic acids, 26 terpenoids and 11 phenols, were identified or tentatively characterized in 30 min. Among them, 49 compounds, including 5 phenylethanoid glycosides, 12 flavonoids, 16 organic acids, 12 terpenoids, and 4 phenols, were identified in Callicarpa kwangtungensis Chun for the first time. Besides, the fragmentation pathways were also discussed. This research established a rapid and reliable method to analyze the chemical compositions of complicated herb without the process of isolation, and provide abundant information on the chemical material basis for further bioactivity and quality control studies.     

Qualitative and quantitative analysis of cinobufacini injection using rapid separation liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry and HPLC‐photodiode array detection,a feasible strategy for the quality control of Chinese medicine injections

Cinobufacini injection, prepared from the skin of Bufo bufo gargarizans Cantor, has presented its significant effects on the treatment of hepatitis and various cancers in the clinic. However, as an unclear complex chemical system, the optimization of its quality control markers has been a long‐term challenge. In present study, a feasible strategy integrated markers screening, determination, and statistical analysis was efficiently proposed, especially for the undefined Chinese medicine injections. First, rapid separation LC‐quadrupole‐TOF‐MS method was applied in the identification of 19 major compounds in the cinobufacini injection for the first time. Further, nine high‐level contents active compounds were selected as quality control markers for the quantification analysis. An acceptable and validated determination method was established in 17 batches of cinobufacini injection by HPLC‐photodiode array detection method, including linear regression relationship (r², 0.9996–1), precisions (RSD, 0.02–1.35%), repeatability (RSD, 0.05–1.97%), stability (RSD, 0.1–3.85%), and recovery (95.88–104.89%). Each analyte was detected at its maximum ultraviolet spectra wavelength. Finally, based on the quantification results, principal component analysis was performed on the quality assessment of cinobufacini injections. This three‐step strategy provides a newly feasible solution for the quality control of Chinese medicine injections.     

Characterization of chemical constituents in Zhi–Zi–Da–Huang decoction by ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

A sensitive and reliable ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was established to separate and identify the chemical constituents of Zhi–Zi–Da–Huang decoction, a classic traditional Chinese medicine formula. The chromatographic separation was achieved on a Shim‐pack XR‐ODS C₁₈ column (75  × 3.0 mm, 2.2 μm) using a gradient elution program. The detection was performed on a Waters Xevo G2 Q‐TOF mass spectrometer equipped with electrospray ionization source in both positive and negative modes. With the optimized conditions, a total of 82 compounds were identified or tentatively characterized. Of the 82 compounds, 21 compounds were identified by comparing the retention time and MS data with reference standards, the rest were characterized by analyzing MS data and retrieving the reference literature. In addition, 31 compounds were identified from Gardenia jasminoides Ellis, ten compounds were identified from Rheum palmatum L., 33 compounds were identified from Citrus aurantium L., and eight compounds were identified from Sojae Semen Praeparatum. Results indicated that iridoids, anthraquinones, flavonoids, isoflavonoids, coumarins, glycosides of crocetin, monoterpenoids, and organic acids were major constituents in Zhi–Zi–Da–Huang decoction. It is concluded that the developed ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method with high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents of Zhi–Zi–Da–Huang decoction, and the analysis provides a helpful chemical basis for further research on Zhi–Zi–Da–Huang decoction.     

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.     

Identification of chemical constituents in traditional Chinese medicine formula using HPLC coupled with linear ion trap‐Orbitrap MS from high doses of medicinal materials to equivalent doses of formula: Study on Xiang‐Sha‐Liu‐Jun‐Zi‐Jia‐Jian granules

High‐resolution mass spectrometry has been a powerful tool for the research of chemical constituents in traditional Chinese medicine (TCM) formulas. However, the chromatographic peaks were difficult to discriminate clearly in data collection or analysis because of the complexity and the greatly different content of the constituents in TCM formula, which increased the difficulty of identification. In this study, a high‐performance liquid chromatography coupled with linear ion trap‐Orbitrap mass spectrometry based strategy focused on the comprehensive identification of TCM formula constituents was developed. Identification was carried out from a high dose of medicinal materials to equivalent dose of formula. Meanwhile, combined with mass spectrometry data, chromatographic behaviors, reference standards and previous reports, the identification of constituents in Xiang‐Sha‐Liu‐Jun‐Zi‐Jia‐Jian granules was described. 169 compounds were unambiguously or tentatively characterized, mainly including flavonoids, alkaloids, triterpenic acids, triterpene saponins, lactones, sesquiterpenoids and some other compounds. Among them, 11 compounds were unambiguously confirmed by comparing with reference standards. These results demonstrated that the method was effective and reliable for comprehensive identification of constituents of Xiang‐Sha‐Liu‐Jun‐Zi‐Jia‐Jian granules extracts and reveal the material basis of its therapeutic effects. This strategy might propose a research idea for the characterization of multi‐constituents in TCM formula.     

Rapid characterization of chemical constituents and metabolites of Qi‐Jing‐Sheng‐Bai granule by using UHPLC–Q‐TOF‐MS

Qi‐Jing‐Sheng‐Bai granule is an effective traditional Chinese medicine formula that has been widely used for the treatment of leukopenia post radiotherapy or chemotherapy. However, its chemical constituents were still unclear, which hindered interpreting bioactive constituents and studying integrative mechanisms. In this study, we developed a three‐step strategy to characterize the chemical constituents and metabolites of Qi‐Jing‐Sheng‐Bai by using ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. As a result, a total of 143 compounds, including 56 flavonoids, 51 saponins, and 36 other compounds, of which contained six pairs of isomers, were tentatively identified and characterized via reference standards and by comparing mass spectrometry data with literature. After oral administration of 15 g/kg Qi‐Jing‐Sheng‐Bai, a number of 42 compounds including 24 prototype compounds and 18 metabolites have been detected in the serum of rats. This work serves as the first reference for Qi‐Jing‐Sheng‐Bai chemical components and metabolites. Moreover, it provided a rapid and valid analytical strategy for characterization of the chemical compounds and metabolites of traditional Chinese medicine formula.     

Qualitative and quantitative evaluation of Oroxylum indicum (L.) Kurz by HPLC and LC‐qTOF‐MS/MS

Oroxylum indicum, as a popular functional Chinese herbal medicine for reducing hyperactivity, relieving sore throat, smoothing the liver and adjusting stomach, mainly contains flavonoids. In this study, we aimed to establish a fast and sensitive method that enables to analyze the chemical components in O. indicum qualitatively and quantitatively. First, a total of 42 components were characterized by LC‐quadrupole time‐of‐flight (qTOF)‐tandem mass spectrometry (MS/MS), including 23 flavonoid glycosides, 13 flavonoids and six other types of compounds. Then, 17 characteristic components of the 19 common peaks in the chromatographic fingerprints of O. indicum were confirmed. Fifty samples were classified into two groups by hierarchical clustering analysis and orthogonal partial least squares‐discriminant analysis, which also identified the 10 main chemical markers responsible for differences between samples. Last, the quantitative analysis of multiple components with a single marker method was established for simultaneous determination of six main active components in O. indicum by LC‐UV with oroxin B was chosen as internal reference substance. Finally, a rapid and efficient method integrating HPLC with LC‐electrospray ionization‐qTOF‐MS/MS analysis was established to comprehensively discriminate and assess the quality of O. indicum samples.     

Identification of chemical constituents and rat metabolites of Kangxianling granule by HPLC‐Q‐TOF‐MS/MS

A high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass tandem mass spectrometry method was established to characterize the chemical constituents of Kangxianling granule (KXL), a traditional Chinese medicine formula, and the metabolic profile in rat urine and plasma after oral administration of KXL. A total of 27 compounds in KXL extract and 13 prototype compounds with 12 metabolites in rat urine and plasma were identified. Among the 27 detected compounds, 15 were identified by comparing the retention time and MS data with that of reference compounds and the other 12 compounds were tentatively assigned based on the MS data and reference literature. The main prototype components absorbed in rat were amygdalin, salvianolic acid B, tanshinones and anthraquinones. Hydroxylation, glucuronidation and sulfation were the principal metabolic pathways in rat. The results revealed that the 25 compounds identified in rat urine and plasma were the potential active ingredients of KXL, which provides helpful chemical information for further study of the pharmacology mechanism of KXL. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid characterization of the constituents in Jigucao capsule using ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry

Jigucao capsule is a well-known Chinese patent medicine for the treatment of acute and chronic hepatitis and cholecystitis. The chemical components of Jigucao capsule were not clear resulting from the paucity of relevant studies, which hindered the research of the pharmacological mechanism, the comprehensive development, and utilization of Jigucao capsule in clinical studies. By establishing a high-throughput ultra-performance liquid chromatography quadrupole time of flight mass spectrometry in combination with intelligent UNIFI software data processing platform to automatically characterize and identify the chemical profile of Jigucao capsule, 144 compounds were determined rapidly, including 34 terpenoids, 25 flavonoids, 22 steroids, 21 phenylpropanoids, 10 glycosides, six alkaloids, 13 organic acids, and other 13 components. These compounds may be the active components of Jigucao capsule. In this study, a rapid and robust method for comprehensively analyzing the chemical composition of Jigucao capsule was described and established for the first time. The results will provide a reference for the quality control of Jigucao capsule and the establishment of a higher quality standard, as well as for the pharmacodynamic material basis research.     

Characterization of chemical constituents and absorbed components,screening the active components of gelanxinning capsule and an evaluation of therapeutic effects by ultra‐high performance liquid chromatography with quadrupole time of flight mass spectrometry

We revealed the potential biomarker and pathway of gelanxinning capsule on rat model with coronary heart disease, which aims to clarify holistic therapeutic effect and predict quality‐markers of gelanxinning capsule. Ultra‐high performance liquid chromatography coupled with mass spectrometry based on metabolomics technique was used to find the biomarkers and related metabolic pathways of coronary heart disease model, which evaluates the intervention effect of gelanxinning capsule. Using serum pharmacochemistry of traditional Chinese medicine and Pearson correlation analysis, effective ingredients in serum is analyzed to characterize the activity of gelanxinning capsule on coronary heart disease under valid state. A total of 20 biomarkers from coronary heart disease were identified and 12 of them were regulated by gelanxinning capsule treatment, which is mainly involved in sphingolipid metabolism and glycerophospholipid metabolism. With the high sensitivity liquid chromatography coupled with mass spectrometry technology, a total of 46 compounds from gelanxinning capsule were identified in vitro and 25 of them were absorbed in blood. The correlation analysis of serum biomarkers and absorbed components was used to find 11 compounds as quality‐markers to be responsible for the efficacy of gelanxinning capsule. This strategy was successfully applied to screening of potential mechanism and quality‐markers from herbal medicine.      

Chemical profiling and quantification of ShenKang injection,a systematic quality control strategy using ultra high performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high‐resolution accurate mass spectrometry

ShenKang injection is traditional Chinese medicine used to treat chronic renal failure in China. It is a compound preparation that consists of four herbs: Rhubarb, Salvia miltiorrhiza, Safflower and Radix Astragali . We developed an ultra high pressure liquid chromatography coupled with Q Exactive hybrid quadrupole‐orbitrap high resolution accurate mass spectrometry tandem mass spectrometry method to analyze its chemical compositions, and a total of 90 compounds were identified from ShenKang injection. Among them, 19 major compounds were unambiguously detected by comparing with reference standards. Meanwhile, 13 representative compounds selected as quality control markers were simultaneously quantified in ShenKang injection samples. Chromatographic separation was accomplished on a Waters ACQUITY HPLC® HSS C₁₈ column using gradient elution. The method was validated with respect to linearity, sensitivity, accuracy and precision, reproducibility and stability. And the validated method was successfully applied for simultaneous determination of 13 bioactive compounds in ShenKang injection from ten batches of samples by liquid chromatography with mass spectrometry. The results were analyzed by principal components analysis method, and three compounds had a significant relationship with the quality control of ShenKang injection. This research established a rapid and reliable method for the integrating quality control, including qualitation and quantification of ShenKang injection.     

Systematic chemical profiling of a multicomponent Chinese herbal formula Huo Luo Xiao Ling Dan by ultra high performance liquid chromatography coupled with electrospray ionization quadrupoletime‐of‐flight mass spectrometry

Huo Luo Xiao Ling Dan, a Chinese herbal formula consisting of 11 different herbs, has been used in folk medicine for the treatment of arthritis and other chronic inflammatory diseases. However, the chemical compositions of Huo Luo Xiao Ling Dan are not completely characterized. In the present study, an ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry method in positive and negative ion modes was employed to identify biochemical constitutes in Huo Luo Xiao Ling Dan. As a result, a total of 76 compounds including alkaloids, monoterpene glycosides, iridoids, phenolic acids, and tanshinones, coumarins, lactones, flavones, and their glycosides, triterpenes, and triterpene saponins were characterized by comparing the retention time and mass spectrometry data with reference standards within 5 ppm error or by reference to the reference literature. These results would provide the basis for a further in vivo study of Huo Luo Xiao Ling Dan and information for potential new drug candidates for treating arthritis and other chronic inflammatory diseases.     

Qualitative screening of absorbed indoloquinazoline alkaloids and their metabolites in rat plasma after the oral administration of Wu‐Zhu‐Yu decoction by high‐resolution mass spectrometry with multiple data mining algorithms

A rapid and sensitive liquid chromatography with high‐resolution mass spectrometry method with multiple data processing algorithms was developed and applied for the metabolite profiling of evodiamine and its analogous alkaloids in rat plasma after the administration of Wu‐Zhu‐Yu decoction. All samples were purified using hydrophilic‐lipophilic balanced solid‐phase extraction cartridges and analyzed by a Sciex TripleTOF 5600⁺ mass spectrometer with a 35 min liquid chromatography gradient elution. High‐resolution full‐scan mass spectrometry and information‐dependent acquisition tandem mass spectrometry data were analyzed using multiple data processing approaches. The results indicated that the detected eight prototype alkaloids could be metabolized to 58 metabolites through both phase I and phase II reactions. Oxidation was demonstrated to be the principle metabolic pathway of the parent compounds. The study contributes to the understanding of the absorption and metabolism of the alkaloids in Wu‐Zhu‐Yu decoction and provides a detailed analysis of scientific data.     

Qualitative and quantitative analysis of major constituents from Dazhu Hongjingtian capsule by UPLC/Q‐TOF‐MS/MS combined with UPLC/QQQ‐MS/MS

In this work, a sensitive and efficient method was established and validated for qualitative and quantitative analysis of major bioactive constituents in Dazhu Hongjingtian capsule by liquid chromatography tandem mass spectrometry. A total of 32 compounds were tentatively identified using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Furthermore, 12 constituents, namely gallic acid, 3,4‐dihydroxybenzoic acid, salidroside, p‐ coumaric acid‐4‐O ‐β ‐d ‐glucopyranoside, bergeninum, 4‐hydroxybenzoic acid, 4‐hydroxyphenylacetic acid, syringate, 6′′‐O ‐galloylsalidroside, rhodiosin, rhodionin and kaempferol‐7‐O ‐α ‐l ‐rhamnoside, were simultaneously quantified by the developed ultra‐performance liquid chromatography coupled with a triple quadrupole mass spectrometry method in 9 min. All of them were analyzed on an Agilent ZorBax SB‐C₁₈ column (3.0 × 100 mm, 1.8 μm) with linear gradient elution of methanol–0.1% formic acid water. The proposed method was applied to analyze three batches of samples with acceptable linearity (R , 0.9979–0.9997), precision (RSD, 1.3–4.7%), repeatability (RSD, 1.7–4.9%), stability (RSD, 2.2–4.9%) and recovery (RSD, 0.6–4.4%) of the 12 compounds. As a result, the analytical method possessing high throughput and sensitivity is suitable for the quality control of Dazhu Hongjingtian capsule.     

Rapid simultaneous determination of twelve major components in Pien Tze Huang by ultra‐performance liquid chromatography coupled with triple quadrupole mass spectrometry

An efficient method using ultra‐performance LC coupled with triple quadrupole MS was developed for the rapid determination of 12 major active components in Pien Tze Huang (PZH), a well‐known traditional Chinese formula. Chromatographic separation was achieved on a Waters XBridge BEH RP18 column (50 mm × 2.1 mm id, 1.7 μm) with a gradient mobile phase (A: 0.1% aqueous formic acid and B: acetonitrile with 0.1% formic acid) at a flow rate of 0.8 mL/min. The chromatographic peaks of 12 components were identified by comparing their retention time and MS data with the related reference compounds. Multiple‐reaction monitoring was employed for the quantitative analysis. Ten batches of PZH were analyzed with a good linear regression relationship (r, 0.9987–0.9995), intraday precisions (RSD, 2.05–4.80%), interday precisions (RSD, 1.99–4.98%), repeatability (RSD, 2.21–4.20%), stability (RSD, 3.52–4.81%), and recovery (95.63–104.80%). By using this established method, the present study offered highly sensitive, specific, and speedy determination of 12 major components, which promoted the quality control investigation of PZH greatly.