Identification and determination of chemical constituents of Citrus reticulata semen through ultra high performance liquid chromatography combined with Q Exactive Orbitrap tandem mass spectrometry

Citrus reticulata semen, a traditional Chinese medicinal material, has desirable medicinal and dietary properties. In this study, a method combining ultra high performance liquid chromatography with Q Exactive Orbitrap tandem mass spectrometry was established and validated for the identification and analysis of the chemical components of C. reticulata semen for the first time. The evaluation of different retention times and fragmentation characteristics, as well as comparative analysis with the literature, resulted in the identification of 35 chemical constituents, including 21 flavonoids and 14 other compounds. The 21 flavonoids derived from C. reticulata semen were reported for the first time. Seven of the chemical components of C. reticulata semen were quantitatively analyzed using the developed method under the optimal conditions. The results showed that the content of limonin, hesperidin, nobiletin, synephrine, tangeretin, 3,5,6,7,8,3′,4′‐heptamethoxyflavone and 5‐hydroxide‐6,7,8,3′,4′‐pentamethoxyflavone in C. reticulata semen was 11.1666, 0.0404, 0.0092, 0.0255, 0.0087, 0.0010, and 0.0008 mg/g, respectively. This study demonstrated that the ultra high performance liquid chromatography Q Exactive Orbitrap mass spectrometry based method can be used to rapidly and reliably analyze the chemical constituents of C. reticulata semen. These results provide a scientific basis for further studies of C. reticulata semen.     

Simultaneous determination of eight bioactive components in rat plasma after oral administration of Yan‐Ke‐Ning‐Tablet by liquid chromatography coupled with Q‐Exactive Orbitrap tandem mass spectrometry

A rapid, sensitive and reliable quantitative method based on ultra‐high performance liquid chromatography coupled with Q‐Exactive Orbitrap tandem mass spectrometry was developed for simultaneous determination of berberine, berberrubine, palmatine, jatrorrhizine, columbamine, baicalin, baicalein and wogonin in rat plasma after oral administration with Yan‐Ke‐Ning‐Tablet (YKNT). After precipitation with acetonitrile, the plasma samples were separated on a reverse‐phase C₁₈ column with 1 mm ammonium acetate containing 0.2% acetic acid–acetonitrile as mobile phase. Calibration curves showed good linearity (r > 0.9983) over the tested concentration ranges of 0.5–200 ng/mL for berberine, berberrubine, palmatine, jatrorrhizine and columbamine, and 1–300 ng/mL for baicalin, baicalein and wogonin. The precision (relative standard deviation) at three different concentration levels was <12.15% and the accuracy (relative error) ranged from ?8.24 to 10.85%. No matrix effects were observed with matrix effect value ranging from 89.23 to 107.68%. The extraction recovery was in the range of 82.34–92.31%. The validated assay was further successfully applied to the pharmacokinetic study of these components after oral administration of YKNT. The present study provides the pharmacokinetic profiles of major bioactive components found in YKNT, and provides valuable information regarding the chemical components that were absorbed into plasma, which will be helpful for understanding the therapeutic effects of YKNT.     

Fingerprinting alterations of secondary metabolites of tangerine peels during growth by HPLC-DAD and chemometric methods

Tangerine peels are herbal materials of two coupled traditional Chinese medicines, Pericarpium Citri Reticulatae (PCR) and Pericarpium Citri Reticulatae Viride (PCRV). In this paper, high-performance liquid chromatographic fingerprints of tangerine peels during growth were firstly measured for deliberately collected 34 samples from three species (Citrus reticulata ‘Chachi’, Citrus reticulata ‘Dahongpao’ and Citrus erythrosa Tanaka). After sixteen characteristic components which have similar change trends in the grown process were screened out with the help of heuristic evolving latent projection (HELP) method, score plots of principal component analysis (PCA) successfully presented the grown footprints of tangerine peels. It implied that July might be the best harvest time for PCRV, November and December were better for PCR. Furthermore, hesperidin, nobiletin and tangeretin were screened as chemical markers by loadings of PCA. The HPLC-HELP-PCA strategy has shown its potential in optimization of harvest time and chemical markers’ screening, which will have wide perspective in the analysis of “coupled TCMs”.     

Comparative analysis of volatile constituents in Citrus Reticulata Blanco using GC–MS and alternative moving window factor analysis

The similarities and differences of essential oil components in Album Citri Reticulatae (ACR), Cylindricae Citri Reticulatae (CCR), Folium Citri Reticulatae (FCR), Exocarpium Citri Grandis (ECG), Pericarpium Citri Reticulatae Viride (PCRV) and Pericarpium Citri Reticulatae (PCR) were investigated by GC–MS combined with a chemometric method named alternative moving window factor analysis (AMWFA). And temperature‐programmed retention indices (PTRIs) were used together with mass spectra for identification of the essential oil components. In essential oils of ACR, CCR, FCR, ECG, PCRV and PCR, 28, 26, 61, 62, 52 and 48 components were determined representing 93.13, 94.44, 93.53, 87.67, 99.03 and 98.03% of the total relative content, respectively. Also, the essential oils significantly differed both qualitatively and quantitatively. There were 14 common components among ACR, CCR, FCR, ECG, PCRV and PCR, their abundance varied in the ranges from 32.39% in FCR to 94.66% in PCRV. The results obtained may be helpful to the further study of pharmacological activity for their potential utilization as therapeutical agents.     

Characterization of the metabolite of AdipoRon in rat and human liver microsomes by ultra‐high‐performance liquid chromatography combined with Q‐Exactive Orbitrap tandem mass spectrometry

AdipoRon is an orally active adiponectin receptor agonist. The aim of this study was to characterize the metabolites of AdipoRon in rat and human liver microsomes using ultra‐high performance liquid chromatography combined with Q‐Exactive Orbitrap tandem mass spectrometry (UPLC‐Q‐Exactive‐Orbitrap‐MS) together with data processing techniques including extracted ion chromatograms and a mass defect filter. AdipoRon (10 μm ) was incubated with liver microsomes in the presence of NADPH and this resulted in a total of 11 metabolites being detected. The identities of these metabolites were characterized by comparing their accurate masses and fragment ions as well as their retention times with those of AdipoRon using MetWorks software. Metabolites M1–M3, M6, and M8–M11 were identified for the first time. Metabolite M4, the major metabolite both in rat and human liver microsomes, was further confirmed using the reference standard. Our results revealed that the metabolic pathways of AdipoRon in liver microsomes were N‐dealkylation (M2), hydroxylation (M, M5–M9), carbonyl reduction (M4) and the formation of amide (M10 and M11). Our results provide valuable information about the in vitro metabolism of AdipoRon, which would be helpful for us to understand the mechanism of the elimination of AdipoRon and, in turn, its effectiveness and toxicity.     

A novel liquid chromatography Orbitrap mass spectrometry method with full scan for simultaneous determination of multiple bioactive constituents of Shenkang injection in rat tissues: Application to tissue distribution and pharmacokinetic studies

Shenkang injection is a traditional Chinese formula with good curative effect on chronic renal failure. In this paper, a novel, rapid and sensitive ultra‐high‐performance liquid chromatography coupled with Q Exactive hybrid quadrupole Orbitrap high‐resolution accurate mass spectrometry was developed and validated for simultaneous determination of seven bioactive constituents of Shenkang injection in rat plasma and tissues after intravenous administration. Acetonitrile was used as a protein precipitation agent in biological samples disposal with carbamazepine as internal standard. The chromatographic separation was carried out on a C₁₈ column with a gradient mobile phase consisting of acetonitrile and water (containing 0.1% formic acid). The MS analysis was performed in the full‐scan positive and negative ion mode. The lower limits of quantification for the seven analytes in rat plasma and tissues were 0.1–10 ng/mL. The validated method was successfully applied to tissue distribution and pharmacokinetic studies of Shenkang injection after intravenous administration. The results of the tissue distribution study showed that the high concentrations of seven constituents were primarily in the kidney tract. This is the first report of the application of Q‐Orbitrap with full‐scan mass spectrometry in tissue distribution and pharmacokinetic studies of Shenkang injection.     

Chemical fingerprint of Ganmaoling granule by double‐wavelength ultra high performance liquid chromatography and ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A method incorporating double‐wavelength ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was developed for the investigation of the chemical fingerprint of Ganmaoling granule. The chromatographic separations were performed on an ACQUITY UPLC HSS C₁₈ column (2.1 × 50 mm, 1.8 μm) at 30°C using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. A total of 11 chemical constituents of Ganmaoling granule were identified from their molecular weight, UV spectra, tandem mass spectrometry data, and retention behavior by comparing the results with those of the reference standards or literature. And 25 peaks were selected as the common peaks for fingerprint analysis to evaluate the similarities among 25 batches of Ganmaoling granule. The results of principal component analysis and orthogonal projection to latent structures discriminant analysis showed that the important chemical markers that could distinguish the different batches were revealed as 4,5‐di‐O‐caffeoylquinic acid, 3,5‐di‐O‐caffeoylquinic acid, and 4‐O‐caffeoylquinic acid. This is the first report of the ultra high performance liquid chromatography chemical fingerprint and component identification of Ganmaoling granule, which could lay a foundation for further studies of Ganmaoling granule.     

Rapid separation and identification of major constituents in Pseudolarix kaempferi by ultra‐performance liquid chromatography coupled with electrospray and quadrupole time‐of‐flight tandem mass spectrometry

A rapid and reliable method based on ultra‐performance liquid chromatography (UPLC) coupled with photodiode‐array detection (PDA) and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (ESI‐Q‐TOF‐MS/MS) has been developed for separation and identification of major constituents in extracts of root bark of Pseudolarix kaempferi Gordon (PKG). Identification of the constituents was carried out by interpretation of their retention times, UV absorption spectra, MS and MS/MS spectra, as well as the data provided by authentic standards and literatures. A total of 20 components were separated in only 8.0 min on a small particle size C₁₈ column (1.7 µm). These components included nine diterpene acids, seven glycosides and four triterpenoids, among which pseudolaric acid C‐O‐β‐D‐glucopyranoside and pseudolaric acid C₂‐O‐β‐D‐glucopyranoside were separated and identified for the first time in this study. Furthermore, the fragmentation patterns of the three types of compounds were elucidated for the first time. This established UPLC‐PDA/Q‐TOF‐MS/MS method is reliable and effective for the separation and identification of the 20 compounds and will be useful for quality control of the crude materials of Pseudolarix kaempferi Gordon and their related preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Efficient analysis of phytochemical constituents in the peel of Chinese wild citrus Mangshanju (Citrus reticulata Blanco) by ultra high performance liquid chromatography–quadrupole time‐of‐flight‐mass spectrometry

An efficient ultra high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was developed for separation and profiling of phytochemical constituents of Chinese wild mandarin Mangshanju (Citrus reticulata Blanco). All constituents were well separated within 16 min. Based on retention times, accurate mass, MS^E fragments, and/or reference standards as well as databases, a total of 81 compounds were unambiguously identified or tentatively assigned including flavonoid glycosides, acylated flavonoid glycosides, flavones, polymethoxylated flavonoids, and limonoids as well as four other compounds. Among them, 22 polymethoxylated flavones and ten polymethoxylated flavanones/chalcones were identified in Mangshanju, more types than other citrus reported before. A basic procedure for identifying flavonoid‐O‐glycosides and the aglycones including polymethoxylated flavonoids was proposed. In addition, this method was successfully used to analyze another four mandarin germplasms, Cenxi suan ju, Xipi gousi gan, Nanfeng miju, and Or, showing that Mangshanju contained two characteristic compounds distinct from the other four citrus species. This study systematically profiled phytochemical constituents of Mangshanju, which was helpful for further utilization of Mangshanju owing to its abundant bioactive compounds.     

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.     

Development and validation of a simple and sensitive high‐resolution LC/MS method for determination of PF‐04620110 in dog plasma: Application to a pharmacokinetic study

In this study, a more sensitive and reliable quantitative method based on ultra‐high performance liquid chromatography coupled with Q‐Exactive‐Orbitrap‐MS in full‐mass scan was developed and validated for the determination of PF‐04620110 in dog plasma. After protein precipitation with acetonitrile, the sample separations were carried out on an Acquity BEH C₁₈ column with 1 mm ammonium acetate in water and acetonitrile containing 0.1% acetic acid as mobile phase, at a flow rate of 0.4 mL/min. The assay showed excellent linearity over the concentration range of 1–2000 ng/mL with correlation coefficient >0.9980 (r > 0.9980). The LLOQ was 1 ng/mL. The inter‐ and intra‐day precision (RSD, %) was within 9.69% while the accuracy (RE, %) was in the range of ?8.59–11.24%. The extraction recovery was >85.37% and the assay was free of matrix effects. PF‐04620110 was demonstrated to be stable under various processing and handing conditions. The validated method was successfully applied to the pharmacokinetic study of PF‐04620110 in dogs and the results revealed that PF‐04620110 was slowly eliminated from plasma with a clearance of 60.81 ± 7.11 mL/h/kg for intravenous administration and 81.44 ± 25.79 mL/h/kg for oral administration. The oral bioavailability was determined to be 77.89% in dogs.     

Qualitative and quantitative determination of Atractylodes rhizome using ultra‐performance liquid chromatography coupled with linear ion trap–Orbitrap mass spectrometry with data‐dependent processing

A quick and effective workflow based on ultra‐performance liquid chromatography coupled with electron spray ionization and LTQ‐Orbitrap mass spectrometry (UPLC‐LTQ‐Orbitrap MS) was established for compositional analysis and screening of the characteristic compounds of three species of Atractylodes rhizome for quality evaluation. This technique was employed to determine the seven main components in Atractylodes rhizome samples. Ultimately, 78 constituents were identified; of these, seven characteristic compounds were selected for species discrimination, comprising atractylodin (63), atractylenolide I (43), atractylenolide II (49), atractylenolide III (53), atractylon (69), methyl‐atractylenolide II (54) and (4E,6E,12E)‐tetradecadecatriene‐8,10‐diyne‐1,3‐diacetate (59). The seven main compounds, including six characteristic compounds, were simultaneously determined in 29 batches of Atractylodes rhizome samples. Thus, the method validation showed acceptable results. Quantitative analysis showed significantly different contents of the seven main components among the three species of Atractylodes rhizome, which indicates possible distinctions in the pharmacological effects. This established method can simultaneously provide qualitative and quantitative results for compositional characterization of Atractylodes rhizomes and for quality control.     

Rapid discovery of absorbed constituents and metabolites in rat plasma after the oral administration of Zi Shen Wan using high‐throughput UHPLC–MS with a multivariate analysis approach

Zi Shen Wan is a typical formula consisting of three herbs, Phellodendri Amurensis Cortex, Rhizoma Anemarrhenae, and Cortex Cinnamomi, and has been widely used for treating prostatitis and infection diseases. However, it lacks in‐depth research of the constituents of Zi Shen Wan in vivo and in vitro. In this work, ultra high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry and MassLynx software was established to characterize the chemical compositions of Zi Shen Wan in vivo and in vitro. In total, 92 peaks were characterized in vitro and 33 peaks were characterized in vivo based on mass spectrometry and tandem mass spectrometry data. Among the 33 compounds characterized in rat plasma, 22 prototype components absorbed in rat serum and 11 metabolites were identified in vivo. This work was fully reports the chemical constituents of traditional Chinese formula of Zi Shen Wan, it demonstrated that ultra high performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry coupled to MassLynx software and multivariate data processing approach could be successfully applied for rapid screening and comprehensive analysis of chemical constituents in vitro and prototype components or metabolites in vivo of traditional Chinese 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.     

Serum pharmacochemistry combined with multiple data processing approach to screen the bioactive components and their metabolites in Mutan Cortex by ultra‐performance liquid chromatography tandem mass spectrometry

Root cortex of Paeonia suffruticosa Andrews (Paeoniaceae), known as Moutan Cortex (MC), is known to have anti‐allergic and anti‐inflammatory properties. However, the constituents absorbed into blood after oral administration of MC remain unknown. A sensitive and rapid method by ultra‐high‐pressure liquid chromatography–electrospray ionization–quadrupole‐time‐of‐flight mass spectrometry (UPLC‐ESI‐Q‐TOF‐MS) technology and the MetaboLynx^TM software combined with multiple data processing approach (Mdpa) was established to investigate the absorbed constituents in rats after oral administration of MC, providing unique high‐throughput capabilities for drug metabolism study. A hyphenated electrospray ionization and quadrupole‐time‐of‐flight analyzer was used for the determination of accurate mass of the fragment ion in negative mode, with excellent MS mass accuracy and enhanced data acquisition. This rapid automated analysis method was successfully applied for screening and identification of the constituents absorbed and metabolized studies of MC after oral administration to rats. A total of 46 peaks were obtained from MC, 41 of which were tentatively characterized. In the VIP‐plot of orthogonal partial least‐squares discriminant analysis, 23 interesting ions in serum samples were extracted, and 16 parent components and seven metabolites were detected in vivo. The integrative serum pharmacochemistry technique, UPLC‐ESI‐Q‐TOF‐MS, and Mdpa method were successfully applied for rapid discovery of multiple components from MC. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparative analysis of 15 chemical constituents in Scutellaria baicalensis stem‐leaf from different regions in China by ultra‐high performance liquid chromatography with triple quadrupole tandem mass spectrometry

Scutellaria baicalensis is a traditional Chinese herbal medicine containing multiple components, which has been extensively used in clinics to treat epidemic febrile disease and hyperactivity cough. To get a deeper understanding about Scutellaria baicalensis stem‐leaf resources, we analyzed 15 chemical constituents in 35 batches of Scutellaria baicalensis stem‐leaf from eight regions in China. A rapid, simple, and sensitive method using ultra‐high performance liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry has been developed for the first time to simultaneously determine 15 chemical constituents (including phenolic acids and flavonoids) in Scutellaria baicalensis stem‐leaf. Sufficient separation of 15 target constituents was achieved on a Waters Acquity UPLC BEH C₁₈ (2.1 mm × 100 mm, 1.7 μm) column within 14 min under the optimized chromatographic conditions. The established method was validated and showed good linearity, precision, repeatability, stability, and recovery and was successfully applied for the simultaneous determination of the 15 chemical constituents in these samples. Hierarchical clustering analysis and principal components analysis were performed to estimate and classify these samples based on the contents of the 15 chemical constituents. This study provided theoretical basis and scientific evidence for the development and utilization of Scutellaria baicalensis stem‐leaf resources.     

Identification and determination of the major constituents in Kai‐Xin‐San by UPLC‐Q/TOF MS and UFLC‐MS/MS method

In order to have overall chemical material information of Kai‐Xin‐San (KXS), the reliable ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometer (UHPLC–Q‐TOF‐MS) and ultra‐fast liquid chromatography mass spectrometer (UFLC‐MS/MS) methods were developed for the identification and determination of the major constituents in KXS. Moreover, the UHPLC–Q‐TOF‐MS method was also applied to screen for multiple absorbed components in rat plasma after oral administration of KXS. The UHPLC–Q‐TOF‐MS method was achieved on Agilent 6520 Q‐TOF mass and operated in the negative ion mode. Good separation was performed on a ZORBAX Eclipse Plus C18 column with a gradient elution at a flow rate of 0.2 ml/min. A total of 92 compounds in KXS were identified or tentatively characterized based on their exact molecular weights, fragmentation patterns, and literature data. A total of 26 compounds including 23 prototype components and three metabolites were identified in rat plasma after oral administration of KXS. Then, 16 major bioactive constituents were chosen as the benchmark substances to evaluate the quality of KXS. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple‐reaction monitoring mode(MRM). The analysis was completed with a gradient elution at a flow rate of 0.4 ml/min within 35 min. The simple and fast method was validated and showed good linearity, precision, and recovery. Furthermore, the method was successful applied for the determination of 16 compounds in KXS. All results would provide essential data for identification and quality control of active chemical constituents in KXS. Copyright © 2016 John Wiley & Sons, Ltd.     

Qualitative and quantitative determination of YiXinShu Tablet using ultra high performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high‐resolution accurate mass spectrometry

To clarify and quantify the chemical profile of YiXinShu Tablet rapidly, a feasible and accurate strategy was developed by applying ultra high performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high‐resolution accurate mass spectrometry. A total of 105 components were identified, including 25 phenanthraquinones, 11 lactones, 19 lignans, 24 acids, and 26 other compounds. Among them, 26 major compounds were unambiguously detected by comparing with reference standards. And 19 of these compounds in three batches of YiXinShu Tablet were selected for quantitative determination. (Z )‐Ligustilide, salvianic acid A, salvianolic acid A, salvianolic acid B, and rosmarinic acid were abundant in these three batches with contents over 1 mg/g. The established analysis methods were examined to be accurate and feasible. The results show that the ultra high performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high‐resolution accurate mass spectrometry method has a powerful qualitative ability and promising quantitative application.