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.     

Biotransformation and metabolic profile of caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐cymaropyranoside with human intestinal microflora by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In our previous studies, caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐ cymaropyranoside (CDMC) was for the first time isolated from Cynanchum auriculatum Royle ex Wightand and was reported to possess a wide range of biological activities. However, the routes and metabolites of CDMC produced by intestinal bacteria are not well understood. In this study, ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) technique combined with Metabolynx^TMsoftware was applied to analyze metabolites of CDMC by human intestinal bacteria. The incubated samples collected for 48 h in an anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF‐MS within 12 min. Eight metabolites were identified based on MS and MS/MS data. The results indicated that hydrolysis, hydrogenation, demethylation and hydroxylation were the major metabolic pathways of CDMC in vitro. Seven strains of bacteria including Bacillus sp. 46, Enterococcus sp. 30 and sp. 45, Escherichia sp. 49A, sp. 64, sp. 68 and sp. 75 were further identified using 16S rRNA gene sequencing owing to their relatively strong metabolic capacity toward CDMC. The present study provides important information about metabolic routes of CDMC and the roles of different intestinal bacteria in the metabolism of CDMC. Moreover, those metabolites might influence the biological effect of CDMC in vivo, which affects the clinical effects of this medicinal plant. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Identification of bio‐active metabolites of gentiopicroside by UPLC/Q‐TOF MS and NMR

Gentiopicroside (GPS), the main bioactive component in Gentiana scabra Bge., has attracted our attention owing to its high bioactivity, especially the treatment of hepatobiliary disorders. The aglycone form of GPS, a typical secoiridoid glycoside, is considered to be more readily absorbed than its parent drug. This study aimed to identify and characterize the metabolites after GPS incubated with β‐glucosidase in buffer solution at 37°C. Samples of biotransformed solution were collected and analyzed by ultraperformance liquid chromatography (UPLC)/quadrupole–time‐of‐flight mass spectrometry (Q‐TOF MS). A total of four metabolites were detected: two were isolated and elucidated by preparative‐HPLC and NMR techniques, and one of those four is reported for the first time. The mass spectral fragmentation pattern and accurate masses of metabolites were established on the basis of UPLC/Q‐TOF MS analysis. Structure elucidation of metabolites was achieved by comparing their fragmentation pattern with that of the parent drug. A fairly possible metabolic pathway of GPS by β‐glucosidase was proposed. The hepatoprotective activities of metabolites M1 and M2 were investigated and the results showed that their hepatoprotective activities were higher than that of parent drug. Our results provided a meaningful basis for discovering lead compounds from biotransformation related to G. scabra Bge. in traditional Chinese medicine. Copyright © 2013 John Wiley & Sons, Ltd.     

Metabolic profile of phillyrin in rats obtained by UPLC‐Q‐TOF‐MS

Forsythia suspensa Vahl (Oleaceae) is an important original plant in traditional Chinese medicine. The air‐dried fruits of Forsythia suspensa have long been used to relieve respiratory symptoms. Phillyrin is one of the main chemical constituent of Forsythia suspensa. A clear understanding of the metabolism of phillyrin is very important in rational clinical use and pharmacological research. In this study, the metabolism of phillyrin in rat was investigated for the first time using an ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) method. Bile, urine and feces were collected from rats after single‐dose (10 mg/kg) orally administered phillyrin. Liquid–liquid extraction and ultrasonic extraction were used to prepare samples. UPLC‐Q‐TOF‐MS analysis of the phillyrin samples showed that phillyrin was converted to a major metabolite, M26, which underwent deglucosidation, further dehydration and desaturation. A total of 34 metabolites were detected including 30 phase I and four phase II metabolites. The conjugation types and structure skeletons of the metabolites were preliminarily determined. Moreover, 28 new metabolites were reported for the first time. The main biotransformation route of phillyrin was identified as hydrolysis, oxidation and sulfation. These findings enhance our understanding of the metabolism and the real active structures of phillyrin. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Rapid analysis of N‐linked oligosaccharides in glycoproteins (ovalbumin,ribonuclease B and fetuin) by reversed‐phase ultra‐performance liquid chromatography with fluorescence detection and electrospray ionization time‐of‐flight mass spectrometry

Rapid, selective and sensitive determination of N‐linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) was performed by ultra‐performance liquid chromatography (UPLC) with fluorescence (FL) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF‐MS). The asparaginyl‐oligosaccharide moiety was first liberated from each glycoprotein by pronase E (a proteolitic enzyme). The oligosaccharide fractions separated by gel‐permeation chromatography were labeled with 1‐pyrenesulfonyl chloride (PSC, a fluorescence reagent), separated by UPLC in a short run time, and then detected by FL and TOF‐MS. The PSC‐labeled oligosaccharides were selectively identified from the FL detection and then sensitively determined by ESI‐TOF‐MS. As the results, 15, eight and four kinds of N‐linked oligosaccharides were detected from ovalbumin, ribonuclease B and fetuin, respectively. Because the present method is rapid (within 9 min), selective and sensitive (approximate 60 fmol, S/N = 5), the determination of N‐linked oligosaccharides in various glycoproteins seems to be possible. Copyright © 2008 John Wiley & Sons, Ltd.     

Light‐Induced Chiral Iron Copper Selenide Nanoparticles Prevent β‐Amyloidopathy In Vivo

The accumulation and deposition of β‐amyloid (Aβ) plaques in the brain is considered a potential pathogenic mechanism underlying Alzheimer's disease (AD). Chiral l/d ‐Fe_xCu_ySe nanoparticles (NPs) were fabricated that interfer with the self‐assembly of Aβ42 monomers and trigger the Aβ42 fibrils in dense structures to become looser monomers under 808 nm near‐infrared (NIR) illumination. d ‐Fe_xCu_ySe NPs have a much higher affinity for Aβ42 fibrils than l ‐Fe_xCu_ySe NPs and chiral Cu_2?xSe NPs. The chiral Fe_xCu_ySe NPs also generate more reactive oxygen species (ROS) than chiral Cu_2?xSe NPs under NIR‐light irradiation. In living MN9D cells, d ‐NPs attenuate the adhesion of Aβ42 to membranes and neuron loss after NIR treatment within 10 min without the photothermal effect. In‐vivo experiments showed that d ‐Fe_xCu_ySe NPs provide an efficient protection against neuronal damage induced by the deposition of Aβ42 and alleviate symptoms in a mouse model of AD, leading to the recovery of cognitive competence.     

Bioactivity‐based ultra‐performance liquid chromatography‐coupled quadrupole time‐of‐flight mass spectrometry for NF‐κB inhibitors identification in Chinese Medicinal Preparation Bufei Granule

Traditional Chinese medicine (TCM) preparations have become effective treatments for many diseases. However, their active ingredients are still uncertain and difficult to identify. In this study, we propose a strategy that integrates ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) and bioactive (NF‐κB inhibitor) luciferase reporter assay systems for the rapid determination of various anti‐inflammatory compounds of TCM preparations. In this way, Bufei Granule (BFG), a TCM preparation used for the clinical therapy of asthma, was analyzed by the two ways of component identification and activity detection. Potential anti‐inflammatory constituents were screened by NF‐κB activity assay systems and simultaneously identified according to the mass spectrometry data. Three structural types of NF‐κB inhibitors (caffeic acid derivatives, flavonoids and Pentacyclic triterpenes) were characterized. Further cytokine detection confirmed the anti‐inflammatory effects of the potential NF‐κB inhibitors. Compared with conventional chromatographic separation and inhibitory activity detection, integrating UPLC/Q‐TOF‐MS identification and virtual validation was more convenient and more reliable. This strategy clearly demonstrates that MS data‐based fingerprinting is a meaningful tool not only in identifying constituents in complex matrix but also in directly screening for powerful trace ingredients in TCM preparations. Copyright © 2016 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.     

Identification of isoquercitrin metabolites produced by human intestinal bacteria using UPLC‐Q‐TOF/MS

In this paper, ultraperformance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS) and the MetaboLynx? software combined with mass defect filtering were applied to identity the metabolites of isoquercitrin using an intestinal mixture of bacteria and 96 isolated strains from human feces. The human incubated samples collected for 72 h in the anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF/MS within 10 min. The parent compound and five metabolites were identified by eight isolated strains, including Bacillus sp. 17, Veillonella sp. 23 and 32 and Bacteroides sp. 40, 41, 56, 75 and 88 in vitro. The results indicate that quercetin, acetylated isoquercitrin, dehydroxylated isoquercitrin, hydroxylated quercetin and hydroxymethylated quercetin are the major metabolites of isoquercitrin. Furthermore, a possible metabolic pathway for the biotransformation of isoquercitrin was established in intestinal flora. This study will be helpful for understanding the metabolic route of isoquercitrin and the role of different intestinal bacteria in the metabolism of natural compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Relationship between the UPLC‐Q‐TOF‐MS fingerprinted constituents from Daphne genkwa and their anti‐inflammatory,anti‐oxidant activities

Daphne genkwa Sieb.et Zucc. is a well‐known medicinal plant. This study was designed to apply the ultra‐high performance liquid chromatography system to establish a quality control method for D. genkwa. Data revealed that there were 15 common peaks in 10 batches of D. genkwa Sieb. Et Zucc. (Thymelaeaceae) from different provinces of China. On this basis, the fingerprint chromatogram was established to provide references for quality control. Afterwards, the chemical constitutions of these common peaks were analyzed using the UPLC‐Q‐TOF‐MS system and nine of them were identified. In addition, LPS‐stimulated RAW264.7 murine macrophages and DPPH assay were used to study the anti‐inflammatory and anti‐oxidation effects of D. genkwa . Then the fingerprint–efficacy relationships between UPLC fingerprints and pharmacodynamic data were studied with canonical correlation analysis. Analysis results indicated that the anti‐inflammatory and anti‐oxidation effects differed among the 10 D. genkwa samples owing to their inherent differences of chemical compositions. Taken together, this research established a fingerprint–efficacy relationship model of D. genkwa plant by combining the UPLC analytic technique and pharmacological research, which provided references for the detection of the principal components of traditional Chinese medicine on bioactivity.     

Organoplatinum‐Substituted Polyoxometalate Inhibits β‐amyloid Aggregation for Alzheimer's Therapy

Aggregated β‐amyloid (Aβ) is widely considered as a key factor in triggering progressive loss of neuronal function in Alzheimer's disease (AD), so targeting and inhibiting Aβ aggregation has been broadly recognized as an efficient therapeutic strategy for curing AD. Herein, we designed and prepared an organic platinum‐substituted polyoxometalate, (Me₄N)₃[PW₁₁O₄₀(SiC₃H₆NH₂)₂PtCl₂] (abbreviated as Pt^II‐PW₁₁) for inhibiting Aβ₄₂ aggregation. The mechanism of inhibition on Aβ₄₂ aggregation by Pt^II‐PW₁₁ was attributed to the multiple interactions of Pt^II‐PW₁₁ with Aβ₄₂ including coordination interaction of Pt²⁺ in Pt^II‐PW₁₁ with amino group in Aβ₄₂, electrostatic attraction, hydrogen bonding and van der Waals force. In cell‐based assay, Pt^II‐PW₁₁ displayed remarkable neuroprotective effect for Aβ₄₂ aggregation‐induced cytotoxicity, leading to increase of cell viability from 49 % to 67 % at a dosage of 8 μm . More importantly, the Pt^II‐PW₁₁ greatly reduced Aβ deposition and rescued memory loss in APP/PS1 transgenic AD model mice without noticeable cytotoxicity, demonstrating its potential as drugs for AD treatment.     

Rapid determination of oxidized methionine residues in recombinant human basic fibroblast growth factor by ultra‐performance liquid chromatography and electrospray ionization quadrupole time‐of‐flight mass spectrometry with in‐source collision‐induced dissociation

The primary structure of the deteriorated recombinant human basic fibroblast growth factor (rhbFGF) was determined by ultra‐performance liquid chromatography and electrospray ionization quadrupole time‐of‐flight mass spectrometry (UPLC/ESI‐QTOF‐MS) with in‐source collision‐induced dissociation (CID). The rhbFGFs before and after treatment with hydrogen peroxide (H₂O₂) were separated using an ACQUITY UPLC BEH300 C18 column (1.7 µm, 150 mm × 2.1 mm i.d.) with a gradient elution of a mixture of water/acetonitrile containing 0.1% formic acid. The separated proteins were then detected by a SYNAPT? High Definition Mass Spectrometry? system (SYNAPT‐MS). Two methionine (Met) residues in the rhbFGF structure were oxidized to Met‐sulfoxide (Met‐O) in 0.03% H₂O₂ at pH 2.0. As the result, three peaks, except for the peak of rhbFGF, appeared on the chromatogram. The three proteins corresponding to each peak were estimated as the denatured rhbFGFs including the Met‐O residue(s) with TOF‐MS. Furthermore, the position of the Met‐O residue(s) was efficiently identified by UPLC/ESI‐QTOF‐MS using the in‐source CID technique. The proposed method seems to be very useful for the structural elucidation of proteins, because the oxidized Met residues in rhbFGF were easily and rapidly identified. Copyright © 2009 John Wiley & Sons, Ltd.     

Enhanced matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analysis of bacteriophage major capsid proteins with β‐mercaptoethanol pretreatment

Bacteriophage (phage) proteins have been analyzed previously with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). However, analysis of phage major capsid proteins (MCPs) has been limited by the ability to reproducibly generate ions from MCP monomers. While the acidic conditions of MALDI‐TOF MS sample preparation have been shown to aid in disassembly of some phage capsids, many require further treatment to successfully liberate MCP monomers. The findings presented here suggest that β‐mercaptoethanol reduction of the disulfide bonds linking phage MCPs prior to mass spectrometric analysis results in significantly increased MALDI‐TOF MS sensitivity and reproducibility of Yersinia pestis‐specific phage protein profiles. Copyright © 2009 John Wiley & Sons, Ltd.     

Sequence and phosphorylation level determination of two donkey β‐caseins by mass spectrometry

Two coeluting components, with experimentally measured M_r values of 25529 and 24606 Da, were identified by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and mass spectrometric analysis in the dephosphorylated casein fraction of a milk sample collected from an individual donkey belonging to the Ragusano breed of the east of Sicily. By coupling enzymatic digestions, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and RP‐HPLC/nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) analysis, the two proteins were identified as donkey β‐CNs and their sequences characterized completely, using the two known β‐CNs from mare as references. The two donkey β‐CNs, showing a mass difference of 923 Da, differ by the presence of the domain E²⁷SITHINK³⁴ in the full‐length component (M_r 25529 Da). In comparison with the mare's β‐CNs used as reference, they present nine amino acid substitutions: L→S³⁷, R→H⁵², S→N⁸¹, P→V⁸⁴, L→V⁹¹, R→Q²⁰³, P→L/I²⁰⁶, L→F²¹⁰ and A→P²¹⁹. Together, these substitutions account for the increase of 18 Da in the M_r of the donkey β‐CNs with respect to the counterparts from the mare. The molecular mass determination by ESI‐MS for the phosphorylated proteins showed that the full‐length component was composed of highly multi‐phosphorylated isoforms with five to seven phosphate groups. By analogy with the homologous mare's β‐CNs, the full‐length (226 amino acids) β‐CN was termed variant A, whereas the shorter (218 amino acids) β‐CN was termed variant A^Δ5. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of protostane triterpenoids in Alisma orientalis by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

A reliable and sensitive ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) method has been optimized and established for analysis of protostane triterpenoids in a commonly used traditional Chinese herbal medicine Alisma orientalis (Sam.) Juzep. The separation of crude extract of A. orientalis was achieved on a Waters ACQUITY HSS T3 column (100 mm × 2.1 mm, 1.8 µm) eluting with 0.1% (v/v) formic acid/acetonitrile. A total of 20 protostane triterpenoids including 19 known compounds and a new one were well separated within 7 min. The collision‐induced dissociation (CID) tandem mass spectrometric (MS/MS) fragmentation patterns of protostane triterpenoids was firstly reported in this study. The hydrogen rearrangement at the C‐23‐OH leads to dissociation of the bond between C‐23 and C‐24 in the protostane triterpenoid skeleton during the CID process. This dissociation was the characteristic CID fragmentation pathway of this class of triterpenoids, and was useful for further differentiation of some positional isomers which contain an acetyl unit on the C‐23 or C‐24 position. The identities of isolated compounds were identified by comparing their retention times and CID fragmentation behaviors with those of reference standards or tentatively assigned by matching the empirical molecular formulae with those reported in the literature. It is concluded that this newly established UPLC/Q‐TOF‐MS method is a powerful approach for structural elucidation of protostane triterpenoids isolated from A. orientalis. Copyright © 2010 John Wiley & Sons, Ltd.