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

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

An effective method for determining the ingredients of Shuanghuanglian formula in blood samples using high‐resolution LC–MS coupled with background subtraction and a multiple data processing approach

Shuanghuanglian formula (SF) is a combination of Flos lonicerae japonicae, Radix scutellariae, and Fructus forsythiae, commonly used to treat viral or bacterial infections. However, the constituents absorbed into the blood after oral administration of SF are difficult to determine and thus remain unclear. Here, we report the application of an accurate background subtraction and multiple data processing approach (Bs‐Mpa) for the comprehensive detection of compounds of SF in vivo. A sensitive and reliable ultra‐performance LC coupled with ESI quadrupole TOF MS (UPLC–ESI‐Q‐TOF‐MS) approach coupled with Bs‐Mpa, which is implemented in the Strip tool from UPLC to remove nonrelated ion signals from accurate mass LC–MS data, was established to characterize the chemical constituents and rat metabolites of SF. In the loading plot of the principal component analysis, 68 ions of interest were extracted from blood samples, among them, 39 absorbed prototype components of SF and 29 metabolites were identified in vivo. It is concluded that the integrative Bs‐Mpa method can be successfully applied for the rapid discovery of multiple components from a traditional Chinese medicine. The above challenge was addressed by using the proposed Bs‐Mpa method and it was particularly suitable for applying to the global characterization of the constituents or metabolites in rat blood after oral administration of other well‐known formulae.     

Pharmacokinetics of 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐D‐glucoside in rat using ultra‐performance LC‐quadrupole TOF‐MS

2,3,5,4′‐Tetrahydroxystilbene‐2‐O‐β‐D‐glucoside (THSG) from Polygoni multiflori has been demonstrated to possess a variety of pharmacological activities, including antioxidant, anti‐inflammatory and hepatoprotective activities. Ultra‐performance LC‐quadrupole TOF‐MS with MS Elevated Energy data collection technique and rapid resolution LC with diode array detection and ESI multistage MSⁿ methods were developed for the pharmacokinetics, tissue distribution, metabolism, and excretion studies of THSG in rats following a single intravenous or oral dose. The three metabolites were identified by rapid resolution LC‐MSⁿ. The concentrations of the THSG in rat plasma, bile, urine, feces, or tissue samples were determined by ultra‐performance LC‐MS. The results showed that THSG was rapidly distributed and eliminated from rat plasma. After the intravenous administration, THSG was mainly distributing in the liver, heart, and lung. For the rat, the major distribution tissues after oral administration were heart, kidney, liver, and lung. There was no long‐term storage of THSG in rat tissues. Total recoveries of THSG within 24 h were low (0.1% in bile, 0.007% in urine, and 0.063% in feces) and THSG was excreted mainly in the forms of metabolites, which may resulted from biotransformation in the liver.     

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.     

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.     

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.     

Rapid identification of primary constituents in parotoid gland secretions of the Australian cane toad using HPLC/MS‐Q‐TOF

Toad parotoid gland secretion or toad venom has in recent years been increasingly shown to possess potentially beneficial pharmacological effects; this speculation has drawn much interest centred on elucidating the chemical basis of its multimodal effects. For this purpose, we explored the use of a rapid and accurate analysis method for systemic investigation of the parotoid gland chemistry, when extracted from Australian cane toads. Full‐scan data of cane toad venom extract was acquired using high‐performance liquid chromatography coupled with a hybrid quadrupole–time of flight mass spectrometry system (HPLC/MS‐Q‐TOF), with multiple ionization sources (ESI and APCI) in positive and negative mixed modes. By measuring the exact mass differences between the theoretical and measured mass of each assumed compound, we confirmed the presence of 12 key constituents. The present results demonstrate that the use of HPLC/MS‐Q‐TOF with multiple ionization sources delivers exemplary selectivity and sensitivity, allowing for the rapid and accurate identification of constituents within cane toad venom. This paves the way for this technique to be used in future routine screening of components within the genus Bufo and for key analytes too, then reliably assessed for any purported beneficial (clinic) properties. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Rapid screening and identification of lycodine‐type alkaloids in Lycopodiaceae and Huperziaceae plants by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Lycodine‐type alkaloids have gained significant interest owing to their unique skeletal characteristics and acetylcholinesterase activity. This study established a rapid and reliable method using ultra‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐ESI‐Q/TOF‐MS/MS) for comprehensive characterization of lycodine‐type alkaloids for the first time. The lycodine‐type alkaloids were detected successfully from Lycopodiastrum casuarinoides, Huperzia serrata and Phlegmarirus carinatus in seven plants of the Lycopodiaceae and Huperziaceae families, based on the established characteristic MS fragmentation of five known alkaloids. Furthermore, a total of 13 lycodine‐type alkaloids were identified, of which three pairs of isomers were structurally characterized and differentiated. This study further improves mass analysis of lycodine‐type alkaloids and demonstrates the superiority of UPLC with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of other trace active compounds. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of the chemical constituents of the different processed products of Anemarrhena asphodeloides Rhizomes by high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this work, high‐performance liquid chromatography (HPLC) coupled with a hybrid quadrupole time of‐flight mass spectrometry (Q‐TOF‐MS/MS) was used to study chemical compositions of different processed products of Rhizoma Anemarrhenae (RA). A Grace Alltima^TM C₁₈ column (250 × 4.6 mm, 5 µm) was used for separation. Mobile phase consisted of 0.1% formic acid and acetonitrile, using gradient elution. ESI‐MS data was acquired in both positive and negative mode. The experiment was established on the basis of a series of reference substances (two xanthone and seven saponins) to qualitatively identify the chemical compounds of different processed products of RA by MS analysis. There was no difference in the type of chemical constituents between different processed products of RA. A total of 25 compounds were identified, including four xanthones, 21 steroidal saponins and eight pairs of isomers. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Ultrasound/microwave‐assisted extraction and comparative analysis of bioactive/toxic indole alkaloids in different medicinal parts of Gelsemium elegans Benth by ultra‐high performance liquid chromatography with MS/MS

Indole alkaloids are the main bioactive/toxic components in Gelsemium elegans Benth. To determine the distribution and contents of indole alkaloids in its different medicinal parts, a novel and rapid method using ultra‐high performance LC (UPLC) with MS/MS has been established and validated with an optimized ultrasound/microwave‐assisted extraction method. Four constituents, namely, humantenidine, humantenmine, gelsemine, and koumine, were simultaneously determined in 6 min. Chromatographic separation was achieved on an ultra‐high performance LC BEH C₁₈ column with a gradient mobile phase consisting of methanol and water (containing 0.1% formic acid both in methanol and water) at a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole electrospray MS/MS by positive ion multiple‐reaction monitoring mode. All the analytes showed good linearity (r ≥ 0.9934) within a concentration range from 0.1–25 μg/mL with a LOQ of 25–50 ng/mL. The overall intra‐ and intervariations of four components were <4.7% with an accuracy of 97.3–101.3%. The analysis results showed that there were remarkable differences in the distribution and contents of four chemical markers in the roots, stems, and leaves of G. elegans Benth. The findings can provide necessary and meaningful information for the rational utilization of its resources.     

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.     

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.     

High‐throughput LC–MS method for the rapid characterization of multiple chemical constituents and metabolites of Da‐Bu‐Yin‐Wan

Traditional Chinese medicine is the clinical experience accumulated by Chinese people against diseases. Da‐Bu‐Yin‐Wan is a famous traditional Chinese medicine formula consisting of Phellodendri amurensis Rupr., Anemarrhenae asphodeloides Bge., Radix Rehmanniae Preparata and Chinemys reevesii . In this study, ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight high‐definition mass spectrometry with the control software of Masslynx (V4.1) was established for comprehensive screening and identification of the chemical constituents and serum metabolites of Da‐Bu‐Yin‐Wan in vivo and in vitro. Consequently, 70 peaks in the methanol extract from Da‐Bu‐Yin‐Wan and 38 peaks absorbed into rat blood were characterized. The 70 constituents in vitro included alkaloids, flavonoids, polysaccharide, limonoids, flavonoid, etc. And the 38 constituents consist of 22 absorbed prototypes and 16 metabolites of Da‐Bu‐Yin‐Wan absorbed in vivo. We fully clarified the chemical constituents of Da‐Bu‐Yin‐Wan and provided a scientific strategy for the screening and characterization of the chemical constituents and metabolites of traditional Chinese medicine in vitro and in vivo.     

Identification of chemical constituents in Rhizoma Paridis Saponins and their oral administration in rat plasma by UPLC/Q‐TOF/MS

On‐line ultra‐performance liquid chromatography (UPLC) coupled with diode‐array detection (UPLC/DAD) and electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF‐MS) were used for separation, identification and structural analyses of saponins in Rhizoma Paridis saponins (RPS) and rat plasma after oral administration of RPS. Thirty steroidal saponins in RPS were identified by comparing their retention time, accurate mass measurement and positive and negative mass spectrometry data with that of reference compounds. The UPLC/Q‐TOF‐MS method was proved to be rapid and efficient in that 30 steroidal saponins, including three kinds of saponins (prototype, pennogenyl and diosgenyl saponins) were tentatively characterized within 6 min. After oral administration of RPS, 21 original saponins were absorbed in RPS‐treated rat plasma. Our results indicated that UPLC/Q‐TOF‐MS is a rapid and effective tool for identification of a series of saponins at trace level. Copyright © 2010 John Wiley & Sons, Ltd.