HPLC‐Q‐TOF‐MS/MS for analysis of major chemical constituents of Yinchen–Zhizi herb pair extract

The Yinchen–Zhizi herb pair (YZHP) consists of Herba Artemisiae Scopariae (Yinchen in Chinese) and Fructus Gardeniae (Zhizi in Chinese), and is mainly used to treat icteric hepatitis, itching skin and eczema. However, the bioactive constituents responsible for the pharmacological effects of YZHP are still unclear to date. In this work, a rapid and sensitive method was established to comprehensively study the constituents in YZHP extract by HPLC‐Q‐TOF MS/MS. The analysis was performed on an HPLC system equipped with an Agilent poroshell 120 EC‐C₁₈ column (100 × 2.1 mm, 2.7 mm) working in a gradient elution program coupled to quadrupole‐time‐of‐flight mass spectrometry operating in the negative ion mode. As a result, a total of 46 compounds including 17 from Herba Artemisiae Scopariae and 36 from Fructus Gardeniae were detected and tentatively identified in YZHP extract by comparing the retention time and mass spectrometry and retrieving the reference literature. More importantly, a series of constituents, such as many iridoid glycosides, were reported for the first time in this formula. The HPLC‐Q‐TOF MS/MS method was developed and utilized successfully to identify the major constituents in YZHP extract and would be helpful for further metabolism and pharmacology research on YZHP. Copyright © 2013 John Wiley & Sons, Ltd.     

Qualitative and quantitative analysis of the major constituents in Acorus tatarinowii Schott by HPLC/ESI‐QTOF‐MS/MS

Acorus tatarinowii Schott (ATS) is a well‐known traditional Chinese medicine (TCM) for the treatment of epilepsy, amnesia and insomnia. In this study, a methodology utilizing high‐performance liquid chromatography (HPLC) coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐QTOF‐MS/MS) was established for the separation and structural identification of the major chemical constituents in ATS for the first time. Overall, 46 major constituents including flavonoid glycosides, phenylpropane derivatives, amides and lignans were identified or tentatively characterized. Seven major constituents, including four phenylpropane derivatives and three lignans, were further quantified as marker substances, which showed good linearity within the test ranges. These results indicated that the developed quantitative method was linear, sensitive, and precise for quality control of ATS. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

HPLC/QTOF‐MS/MS application to investigate phenolic constituents from Ficus pandurata H. aerial roots

Ficus pandurata H. aerial roots are used as a traditional Chinese medicine for the treatment of uarthritis, indigestion and hyperuricemia. However, the bioactive constituents responsible for the pharmacological effects of F. pandurata H. are unclear. A simple and efficient HPLC/QTOF‐MS/MS (high‐performance liquid chromatography/electrospray ionization with quadrupole time‐of‐flight tandem mass spectrometry) method was established to detect and identify active constituents in the n‐butanol extract of F. pandurata H. aerial roots. Chemical constituents were separated and investigated by HPLC/QTOF‐MS/MS in the negative‐ion mode. Thirty‐seven compounds, including hydroxycinnamic acid derivatives, hydroxybenzoic acid derivatives, hydroquinone glycosides, flavonoid glycosides, etc., were identified or tentatively characterized in the n‐butanol extract of F. pandurata H. aerial roots by comparing the UV spectra, accurate mass spectra and fragmentation pathways and retrieving the reference literatures. Moreover, the flavonoid trisaccharides and hydroxybenzoic acid derivatives were tentatively characterized in F. pandurata H. for the first time. The analytical tool used here is very valuable in the rapid separation and identification of the multiple and minor constituents in the n‐butanol extract of F. pandurata H. aerial roots. Copyright © 2014 John Wiley & Sons, Ltd.     

Qualitative and quantitative analysis of the chemical constituents in Mahuang‐Fuzi‐Xixin decoction based on high performance liquid chromatography combined with time‐of‐flight mass spectrometry and triple quadrupole mass spectrometers

High‐performance liquid chromatography coupled with time‐of‐flight mass spectrometry (HPLC‐TOF/MS) and high‐performance liquid chromatography–triple quadrupole mass spectrometry (HPLC‐QQQ/MS/MS) were utilized to clarify the chemical constituents of Mahuang‐Fuzi‐Xixin Decoction. There are 52 compounds, including alkaloids, amino acids and organic acids were identified or tentatively characterized by their characteristic high resolution mass data by HPLC‐QQQ/MS/MS. In the subsequent quantitative analysis, 10 constituents, including methyl ephedrine, aconine, songrine, fuziline, neoline, talatisamine, chasmanine, benzoylmesaconine, benzoylaconine and benzoylhypaconine were simultaneously determined by HPLC‐QQQ/MS/MS with multiple reaction monitoring mode. Satisfactory linearity was achieved with wide linear range and fine determination coefficient (r > 0.9992). The relative standard deviations (RSD) of inter‐ and intra‐day precisions were <3%. This method was also validated by repeatability, stability and recovery with RSD <3% respectively. A highly sensitive and efficient method was established for chemical constituents studying, including identification and quantification of Mahuang‐Fuzi‐Xixin decoction.     

Application of an efficient strategy based on MAE,HPLC‐DAD‐MS/MS and HSCCC for the rapid extraction,identification, separation and purification of flavonoids from Fructus Aurantii Immaturus

This study presents an efficient strategy based on microwave‐assisted extraction (MAE), HPLC‐DAD‐MS/MS and high‐speed counter‐current chromatography (HSCCC) for the rapid extraction, identification, separation and purification of active components from the traditional Chinese medicine Fructus Aurantii Immaturus. An LC‐DAD‐MS/MS method was applied for the screening and structural identification of main components in crude extract, and five components were preliminarily identified as neoeriocitrin, narirutin, naringin, hesperidin and neohesperidin according to their UV and mass spectra. An efficient MAE method for the extraction of the three most abundant components (narirutin, naringin and neohesperidin) was optimized by the combination of univariate and multivariate approaches. The crude extract was then separated and purified by HSCCC and a total of 61.6 mg of narirutin, 207.3 mg of naringin and 159.5 mg of neohesperidin at high purities of 98.1, 97.2 and 99.5%, respectively, were obtained from 1.42 g of crude extract. The recoveries of these compounds were 86, 93 and 89%, respectively. 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.     

HPLC‐ESI‐MS/MS of brain neurotransmitter modulator lobeline and related piperidine alkaloids in Lobelia inflata L

There is a renewed interest in lobelia alkaloids because of their activity on the central nervous system. Lobeline, the most active of them, a nicotinic receptor ligand and neurotransmitter transporter inhibitor, is a candidate pharmacotherapy for metamphetamine abuse. In the present work, high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry in positive ion mode was used for investigating the alkaloid profile in Lobelia inflata L. Chromatographic separations were achieved on a Gemini C6‐phenyl reversed‐phase column providing good peak shape and improved selectivity. Being mostly 2,6‐disubstituted piperidines, lobelia alkaloids presented abundant [M + H]⁺ ions with typical fragmentation. Identification was possible from a few specific ions, especially those resulting from excision of one of the substituents. Based on fragmentation pattern of lobeline as reference compound, 52 alkaloids were identified in the aqueous methanolic extract of L. inflata in contrast to the previously known some 20. Structural variability of these alkaloids identified arises basically from their substituents which can be phenyl‐2‐ketoethyl‐ or phenyl‐2‐hydroxyethyl units as well as their methyl‐, ethyl‐ or propyl‐ homologues attached in different combinations. Several propyl homologue lobelia alkaloids and five hydroxypiperidine derivatives were found in the plant at the first time. In addition to 8‐O‐esters of 2‐monosubstituted piperidine alkaloids previously reported by us in L. inflata, a 3‐hydroxy‐3‐phenylpropanoic acid ester of hydroxyallosedamine ring‐substituted was also identified as a new natural product. High‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry can be successfully applied to Lobeliacae plant samples in the routine screening for new and known bioactive constituents, quality control of the crude drug, lobelia herba, alkaloid production studies, breeding and chemotaxonomy. Copyright © 2015 John Wiley & Sons, Ltd.     

Qualitative analysis of Psoraleae Fructus by HPLC‐DAD/TOF‐MS fingerprint and quantitative analysis of multiple components by single marker

A variety of bioactive substances may account for the recognized efficacy and wide clinical application of Psoraleae Fructus in China. A high‐performance liquid chromatography–diode array detector (HPLC‐DAD) fingerprint method was developed to present the comprehensive phytochemical profile of the crude drug. Thirteen major compounds were separated and identified by HPLC coupled with time‐of‐flight mass spectrometry (HPLC/TOF‐MS), namely psoralenoside (PO), isopsoralenoside (IPO), psoralen (PS), isopsoralen (IPS), neobavaisoflavone (NBF), bavachin (BC), corylin (CN), bavachromene (BCM), psoralidin (PD), isobavachalcone (IBC), bacachinin (BCN), corylifol A (CA) and bakuchiol (BK). Then quantitative analysis of multiple components by single marker (QAMS) was applied in content determination of PO, IPO, PS, IPS, BC, IBC, BCN, CA and BK, with NBF as the internal standard. The calculation results indicated no significant difference from the traditional external standard method (p > 0.05, RSD < 2.62%), suggesting that QAMS is a reliable and convenient method for content determination of multiple chemical compositions, especially when there is a shortage of reference substances. In conclusion, simultaneous qualitative and quantitative analysis of Psoraleae Fructus may be fulfilled through the newly proposed method of QAMS combined with HPLC‐DAD/TOF‐MS fingerprint.     

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 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.     

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.     

Characterization of chemical constituents and rats metabolites of Shuanghua Baihe tablets by HPLC‐Q‐TOF‐MS/MS

A high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry method was established to detect as many constituents in rat biological fluids as possible after oral administration of Shuanghua Baihe tablets (SBT). An Agilent Poroshell 120 EC‐C₁₈ column was adopted to separate the samples, and mass spectra were acquired in positive and negative modes. First, the fingerprints of SBT were established, resulting in 32 components being detected within 40 min. Among these compounds, 12 were tentatively identified by comparing the retention times and mass spectral data with those of reference standards and the reference literature; the other 20 components were tentatively assigned solely based on the MS data. Furthermore, metabolites in rat plasma and urine after oral administration of SBT were also analyzed. A total of 19 compounds were identified, including 13 prototypes and six metabolites through metabolic pathways of demethylation and glucuronide conjugation. Glucuronidated alkaloids were the main constituents in the plasma, and were then excreted from urine. This is the first systematic study on the metabolic profiling of SBT. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of the absorbed components and metabolites of modified Huo Luo Xiao Ling Dan in rat plasma by UHPLC‐Q‐TOF/MS/MS

To reveal the material basis of Huo Luo Xiao Ling Dan (HLXLD), a sensitive and selective ultra‐high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF/MS) method was developed to identify the absorbed components and metabolites in rat plasma after oral administration of HLXLD. The plasma samples were pretreated by liquid–liquid extraction and separated on a Shim‐pack XR‐ODS C₁₈ column (75 × 3.0 mm, 2.2 μm) using a gradient elution program. With the optimized conditions and single sample injection of each positive or negative ion mode, a total of 109 compounds, including 78 prototype compounds and 31 metabolites, were identified or tentatively characterized. The fragmentation patterns of representative compounds were illustrated as well. The results indicated that aromatization and hydration were the main metabolic pathways of lactones and tanshinone‐related metabolites; demethylation and oxidation were the major metabolic pathways of alkaloid‐related compounds; methylation and sulfation were the main metabolic pathways of phenolic acid‐related metabolites. It is concluded the developed UHPLC‐Q‐TOF/MS method with high sensitivity and resolution is suitable for identifying and characterizing the absorbed components and metabolites of HLXLD, and the results will provide essential data for further studying the relationship between the chemical components and pharmacological activity of HLXLD.     

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.     

Detection of flavonoids from Spinacia oleracea leaves using HPLC-ESI-QTOF-MS/MS and UPLC-QqQLIT-MS/MS techniques

Spinacia oleracea L. (Spinach) is a leafy vegetable which is considered to have a high nutritional value. Flavonoids in spinach were reported to act as antimutagenic property. Rapid detection of these flavonoids in Spinach was achieved by using HPLC-ESI-QTOF-MS/MS. Thirty six compounds were tentatively identified based on their retention times, accurate mass and MS/MS spectra. The fragmentation patterns of known compounds were applied to elucidate the structure of their corresponding derivatives having the same basic skeleton. Out of thirty six peaks, three peaks were assigned as patuletin and six peaks were assigned as spinacetin derivatives. Twelve compounds were first time identified following the fragmentation pattern of known compounds. Five of the identified compounds i.e., spinacetin, 5,3′,4′-trihydroxy-3-methoxy-6,7-methylenedioxyflavone, protocatechuic acid, ferulic acid and coumaric acid were simultaneously quantified in spinach leaves by a validated UPLC-ESI-MS/MS method under MRM mode.     

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.