Dimension-Enhanced Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry Combined with Intelligent Peak Annotation for the Rapid Characterization of the Multiple Components from Seeds of Descurainia sophia

The complex composition of herbal metabolites necessitates the development of powerful analytical techniques aimed to identify the bioactive components.The seeds of Descurainia sophia(SDS)are utilized in China as a cough and asthma relieving agent.Herein,a dimension-enhanced integral approach,by combining ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry(UHPLC/IMQTOF-MS)and intelligent peak annotation,was developed to rapidly characterize the multicomponents from SDS.Good chromatographic separation was achieved within 38 min on a UPLC CSH C18(2.1×100 mm,1.7μm)column which was eluted by 0.1%formic acid in water(water phase)and acetonitrile(organic phase).Collision-induced dissociation-MS^(2)data were acquired by the data-independent high-definition MS^(E)(HDMS^(E))in both the negative and positive electrospray ionization modes.A major components knockout strategy was applied to improve the characterization of those minor ingredients by enhancing the injection volume.Moreover,a self-built chemistry library was established,which could be matched by the UNIFI software enabling automatic peak annotation of the obtained HDMS^(E)data.As a result of applying the intelligent peak annotation workflows and further confirmation process,a total of 53 compounds were identified or tentatively characterized from the SDS,including 29 flavonoids,one uridine derivative,four glucosides,one lignin,one phenolic compound,and 17 others.Notably,four-dimensional information related to the structure(e.g.,retention time,collision cross section,MS^(1)and MS^(2)data)was obtained for each component by the developed integral approach,and the results would greatly benefit the quality control of SDS.

Improved preparation and identification of aristolochic acid-DNA adducts by solid-phase extraction with liquid chromatography-tandem mass spectrometry

Aristolochic acid （AA） is a known nephrotoxin and potential carcinogen, which can form covalent DNA adducts after metabolic activation in vivo and in vitro. A simple method for preparation and characterization of aristolochic acid-DNA adducts was developed. Four AA-adducts were synthesized by a direct reaction of AAI/AAII with 2′-deoxynucleosides. The reaction mixture was first cleaned-up and pre-concentrated using solid phase extraction （SPE）, and further purified by a reversed-phase high performance liquid chromatography （HPLC）. By the application of developed SPE procedure, matrices and byproducts in reaction mixture could be greatly reduced and adducts of high purity （more than 94% as indicated by HPLC） were obtained. The purified AA-DNA adducts were identified and characterized with liquid-electrospray ionization-quadrupole-time of flight-mass spectrometry （LC-ESI-Q-TOF-MS/MS） and LC-Diode array detector-fluorescence （LC-DAD-FL） analysis. This work provides a robust tool for possible large-scale preparation of AA-DNA adduct standards, which can promote the further studies on carcinogenic and mutagenic mechanism of aristolochic acids.

Analysis of Pharmacodynamic Substance Basis of Fufang Changtai in Treating Colorectal Cancer by UPLC-Q-TOF-MS Combined with Network Pharmacology

[Objectives] To systematically study the main active components of Fufang Changtai(FFCT) in the treatment of colorectal cancer(CRC), and to explore its mechanism of action. [Methods] The main chemical components of FFCT were analyzed by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) combined with automatic analysis platform, and the main pharmacodynamic substances of FFCT were studied by network pharmacology method and its mechanism of action was explored. The binding degree between the active components and the core targets were verified by molecular docking technology. [Results] A total of 86 compounds were identified from FFCT, among which 26 compounds were Ginsenoside Rg3, Ginsenoside Rb1, Astragaloside III, etc. The key target pathway enrichment analysis showed that FFCT played its role in the treatment of CRC mainly through the PI3K-Akt signaling pathway and MAPK signaling pathway. [Conclusions] This study comprehensively identified the FFCT components. Supplemented by network pharmacology and molecular docking technology, it is expected to provide a scientific theoretical basis and an important reference for FFCT therapeutic components identification, key target verification and mechanism of action in the treatment of CRC.

液相色谱/四极杆-飞行时间质谱测定失忆性贝毒的研究

失忆性贝毒(ASP)是一种80年代在加拿大新发现的毒素,是由大量Pseudonitzschia硅藻属海藻生长产生的软骨藻酸(domoic acid,DA,图1)污染所致,与产生ASP相关的贝类包括贻贝等.……

Rapid Profiling and Characterization of the Multicomponents from the Root and Rhizome of Salvia miltiorrhiza by Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry in Combination with Computational Peak Annotation Workflows

Herbal components characterization represents a challenging task because of the co-existing of multiple classes of naturally occurring compounds with wide spans of polarity,molecular mass,and the ubiquitous isomerism.The root and rhizome of Salvia miltiorrhiza have been utilized as a reputable traditional Chinese medicine Salviae Miltiorrhizae Radix et Rhizoma(Dan-Shen)in the treatment of cardiovascular disease.Herein,a dimensionenhanced ultra-high performance liquid chromatography/ion mobility/quadrupole time-of-flight mass spectrometry approach in combination with intelligent peak annotation workflows was established aimed to rapidly characterize the multicomponents from S.miltiorrhiza.Due to the sufficient optimization,satisfactory chromatography separation was enabled on an HSS T3 column within 33 min using 0.1%formic acid in water(A)and acetonitrile(B)as the mobile phase,while the data-independent HDMS^(E) in both the negative and positive electrospray ionization modes was utilized for the high-coverage MS^(2) data acquisition.Streamlined automatic peak annotation by searching an in-house library(recording 198 known compounds)followed by the subsequent confirming steps(e.g.,comparison with the reference compounds,fragmentation pathways analysis,and retention behavior comparison,etc.),allowed us to identify or tentatively characterize a total of 86 components(including 50 terpenoids,21 phenolic acids,and 15 others)from S.miltiorrhiza.Importantly,three-dimensional structure information,such as the retention time,MS^(1) and MS^(2) data,and collision cross section(CCS),was provided,which can facilitate the more reliable characterization of herbal components.

Characterization and quantitation of aristolochic acid analogs in different parts of Aristolochiae Fructus, using UHPLC-Q/TOF-MS and UHPLC-Qq Q-MS

Aristolochiae Fructus, a Chinese herbal medicine derived from the fruit of Aristolochia contorta Bge., contains nephrotoxic aristolochic acid analogues(AAAs). According to ancient medical texts, various medicinal parts of the fruit of A. contorta were ever used. In order to reveal which part could be safely and effectively used, it is necessary to analyze the chemical profiles of different medicinal parts. Herein we compared the chemical compositions and determined aristolochic acid I(AA-I) and aristolochic acid II(AA-II) in the four parts viz. outer pericarp, inner pericarp, septum, and seed. Ultra-high performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry(UHPLC-QTOF-MS) was applied for chemical profiling. Ultra-high performance liquid coupled with triple quadrupole mass spectrometry(UHPLC-Qq Q-MS) was employed to quantify AA-I and AA-II in different parts. It was found that the chemical compositions of the four parts varied both qualitatively and quantitatively. A total of 10 AAAs, including 5 aristolochic acids and 5 aristolactams, together with 3 alkaloids, were unambiguously or tentatively identified by UHPLC-QTOF-MS. The quantitatively analytical results obtained by UHPLC-Qq Q-MS showed that AA-I and AA-II exclusively accumulate in the seeds of A. contorta. These findings provide supporting data for the rational selection of medicinal parts.

Urinary metabolomics analysis of the anti-depressive effects of Hemerocallis citrina extracts in a simulated microgravity-induced rat model of depression

We investigated the antidepressant-like activity of Hemerocallis citrine Baroni extract(HCE)in a simulated microgravity(SMG)-induced rat model of depression using a metabolomics method.A rat model,generated via 14 d of SMG induction,was validated from the reduced sucrose preference and the enhanced immobility time in the forced swimming test.HCE and paroxetine reversed certain metabolite profiles.Anti-depressant effects of HCE might involve the regulation of several metabolic pathways,such as phenylalanine,glutamic acid,and tryptophan metabolism and changes in energy metabolism.5-Hydroxytryptophan,hippuric acid,phenylacetylglycine,citric acid,3-hydroxykynurenine,cyclic AMP,and L-DOPA profiles were altered upon HCE and paroxetine administration.Furthermore,glutamic acid was only regulated in the HCE group,while xanthurenic acid and deoxyuridine were reversed in the positive group,suggesting differences in the mechanisms between the positive drugs and HCE in improving glutamic acid metabolism.This study provided a theoretical foundation for the application of HCE in depression therapy.

Analysis and Identification of Chemical Constituents of Fenugreek by UPLC-IT-MSn and UPLC-Q-TOF-MS

Fenugreek, a traditional Chinese medical plant, has been widely used as food ingredients because of its out- standing medicinal qualities. The hypoglycemic activity of fenugreek is one of its important pharmacological properties. Both of saponin and flavonoid components have the hypoglycemic activity and their contents in fenugreek are 4%--8% and 1% ---2%, respectively. This paper focused on these two types of components to carry out purification research and chemical analysis. The heating reflux extraction method and macroporous resin purification method were designed to prepare the saponins and the flavonoids components from defatted fenugreek seeds. Petroleum ether ultrasonic skim and 70% ethanol refluxing were used for the extraction of saponins and flavonoid from fenugreek. The column of DM130 macroporous resin and D101 macroporous resin were respectively used to prepare fenugreek saponin and flavonoid components, and the total contents of them were 78.56% and 62.28%, respectively. The saponin and flavonoids were subsequently analyzed by an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry（UPLC-Q-TOF-MS） along with an ultra-performance liquid chromatography and ion-trap tandem mass spectrometry（UPLC-IT-MS＂）. As a result, 57 saponins and 19 flavonoid compounds were characterized. The obtained results will provide a theory basis for further research on fenugreek, as well as research and development of hypogly- cemic new drugs.

Screening Safflower Injection for Constituents with Activity against Stroke Using Comprehensive Chemical Profiling Coupled with Network Pharmacology

Objective:This study aimed to explore safflower injection(SI)for constituents with activity against ischemic stroke using a combination of chemical analysis and a network pharmacology strategy.Materials and Methods:The main ingredients of SI were comprehensively identified using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry,and the core targets and pathways associated with stroke were predicted using PharmMapper and Kyoto Encyclopedia of Genes and Genomes analysis.Cytoscape software was used to visualize and analyze the active compound-target-pathway network of SI regulating ischemic stroke.Results:A total of76 chemical compounds were identified from the SI sample,including 63,which regulated 88 targets that were ultimately enriched in 12 key ischemia stroke-related signaling pathways.Kaempferol-3-O-sophoroside,kaempferol-3-O-rutinoside,carthamoside B6,neoeriocitrin,and6-hydroxykaempferol-3-O-rutinoside-6-O-glucoside were determined to be important for stroke treatment because they had a higher degree value in the network than other constituents did.Moreover,the characteristic components isolated from SI showed protective effect mainly by acting on multiple targets including AKT1,epidermal growth factor receptor,transforming growth factor-beta receptor(TGFBR),Ras homolog,mTORC1 binding,caspase 3,and glycogen synthase kinase 3 beta,which were involved in different signaling pathways including phosphoinositide 3-kinase-Akt,mitogen-activated protein kinase,neurotrophin,ErbB,mechanistic target of rapamycin,and tumor necrosis factor.Conclusions:This study proposed a network pharmacology and chemical component profiling strategy for the systematic understanding of the therapeutic material basis of using SI against ischemic stroke.

Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry and Database-Driven Automatic Peak Annotation for the Rapid Profiling and Characterization of the Multicomponents from Stephaniae Tetrandrae Radix(Fang-Ji)

Objective:Quality control of traditional Chinese medicine(TCM)begins with the chemical basis elucidation.The root of Stephania tetrandra has long been utilized as an antirheumatic,analgesic,and diuretic TCM,Stephaniae Tetrandrae Radix(STR;Fang-Ji).Powerful analytical strategies enabling its multicomponent characterization is still rare.Methods:A rapid,reliable,and enhanced profiling approach,by ultra-high performance liquid chromatography coupled with ion mobility/quadrupole time-of-flight mass spectrometry(UHPLC/IM-QTOF-MS)and automatic peak annotation facilitated by computational matching of in-house library,was established and utilized to characterize the multicomponents from STR.A knockout strategy was utilized by automated valve switching to overcome the interference of predominant peaks.Results:Good chromatographic separation was achieved within 17 min on a reversed-phase BEH C18 column eluted with acetonitrile/0.1%ammonium hydroxide in water,while data-independent high-definition MS^(E)(HDMS^(E))in positive mode was applied to acquire the MS^(2)data by using a Vion TM IM-QTOF instrument,which in theory,could cover all the profiled precursor ions.An in-house library of 163 compounds was established and incorporated into the UNIFITM platform.By feat of these efforts,we were able to identify or tentatively characterize 76 alkaloids from the methanolic extract of STR,including 14 aporphine-type,four morphine-type,48 bisbenzylisoquinoline-type,seven tetrahydroprotoberberine-type,one protopine-type,one benzylisoquinoline-type,and one other.Four-dimensional information,such as the retention time,collision cross section(CCS),high-accuracy MS1 and MS2 data,for each component was provided.Conclusions:The systematic multicomponent characterization of STR was accomplished with high coverage,high degree of automation,and high reliability.