Offline two-dimensional liquid chromatography coupled with ion mobility-quadrupole time-of-flight mass spectrometry enabling fourdimensional separation and characterization of the multicomponents from white ginseng and red ginseng

Inherent complexity of plant metabolites necessitates the use of multi-dimensional information to accomplish comprehensive profiling and confirmative identification.A dimension-enhanced strategy,by offline two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry(2 D-LC/IM-QTOF-MS)enabling four-dimensional separations(2 D-LC,IM,and MS),is proposed.In combination with in-house database-driven automated peak annotation,this strategy was utilized to characterize ginsenosides simultaneously from white ginseng(WG)and red ginseng(RG).An offline 2 DLC system configuring an Xbridge Amide column and an HSS T3 column showed orthogonality 0.76 in the resolution of ginsenosides.Ginsenoside analysis was performed by data-independent high-definition MSE(HDMSE)in the negative ESI mode on a Vion?IMS-QTOF hybrid high-resolution mass spectrometer,which could better resolve ginsenosides than MSEand directly give the CCS information.An in-house ginsenoside database recording 504 known ginsenosides and 58 reference compounds,was established to assist the identification of ginsenosides.Streamlined workflows,by applying UNIFI?to automatedly annotate the HDMSEdata,were proposed.We could separate and characterize 323 ginsenosides(including 286 from WG and 306 from RG),and 125 thereof may have not been isolated from the Panax genus.The established 2 D-LC/IM-QTOF-HDMSEapproach could also act as a magnifier to probe differentiated components between WG and RG.Compared with conventional approaches,this dimensionenhanced strategy could better resolve coeluting herbal components and more efficiently,more reliably identify the multicomponents,which,we believe,offers more possibilities for the systematic exposure and confirmative identification of plant metabolites.

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.

Screening and Identifying of Nephrotoxic Compounds in Lithospermum erythrorhizon Using Live-cell Fluorescence Imaging and Liquid Chromatography Coupled with Tandem Mass Spectrometry

In order to identify the potential nephrotoxic compounds in traditional Chinese medicine Lithospermum erythrorhizon,it was separated into serial fractions according to their polarities.An in vitro method was utilized to determine the nephrotoxicity of these fractions with the help of fluorescence image analysis.As a result,the primary fraction A05 and its secondary fractions C06 ＂C09 and C12 ＂C14 were found to have significant toxicity to LLC-PK1 cell line,as determined by the survive rate less than 20% after they were treated with these fractions.These potential nephrotoxic fractions were further analyzed by multistage and high resolution mass spectrometry.The main compounds in these fractions were tentatively identified to be acetylshikonin,isobutyrylshikonin,β,β＇-dimethyla-cryloylshikonin,and isovalerylshikonin,which may bring nephrotoxicity.

COMPREHENSIVE TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY COUPLED WITH QUADRUPLE TIME-OF-FLIGHT MASS SPECTROMETRY FOR CHEMICAL CONSTITUENTS ANALYSIS OF TRIPTERYGIUM GLYCOSIDES TABLETS

Comprehensive two-dimensional liquid chromatography platform(LC×LC)coupled with quadrupole time-of-flight(QTOF)mass spectrometry(MS)is developed to separate,identify and relatively determine the chemical constituents of two types of tripterygium glycosides tablets(TGT).The types and relative contents of the constituents discovered in two kinds of TGT tablets were subsequently compared.C8andC18 column were used for the separation of the first

Rapid Differentiation of Aconiti Kusnezoffii Radix from Different Geographic Origins Using Ultra-Performance Liquid Chromatography Coupled with Time-of-Flight Mass Spectrometry

Objective:There are different geographic origins of Aconiti Kusnezoffii Radixs(AKRs)sold in the market with different quality.This study aims to establish a rapid analysis method to distinguish the different geographic origins of AKRs and to realize the rapid evaluation of their quality.Methods:An ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry(UPLC-Q-TOF MS)method was utilized to acquire the constituents'information of AKRs from different geographic origins.MSE data and Progenesis QI software were employed to identify the chemical constitutes.Principal component analysis(PCA)was applied to comparing MS data to find the chemical markers of AKRs from different geographic origins.Results:Twenty-three components were detected and 17 out of them were identified,including diester-diterpenoid alkaloids,monoester-diterpenoid alkaloids,and amine-diterpenoid alkaloids.Three pairs of isomers were detected and two of them were distinguished by the retention time of standard samples.Thirteen chemical markers were screened out through PCA and orthogonal partial least square discriminant analysis.Through detecting Napelline or isomer of Napelline(m/z 360.2530)and Aconifine(m/z 662.3170),AKRs from inner Mongolia autonomous could be screened.According to the existence of benzoylaconine(m/z 604.3108)and Indaconitine(m/z 630.3159),it could be confirmed that the AKRs are from Xinjiang Uygur autonomous.AKRs that cannot detect compounds above-mentioned could be from Liaoning or Shanxi Province.Conclusions:The chemical profile could be used not only to distinguish the AKRs from different geographic origins but also to identify the true and false of AKRs.This study lays a foundation for the study of efficacy and toxic of AKRs.

Ultra-performance Liquid Chromatography-Quadrupole/Time-of-Flight Mass Spectrometry Based Bile and Urine Metabonomics Study on the Ameliorative Effects of Curcuma wenyujin Rhizoma on Acute Blood Stasis in Rats

Background: Curcuma wenyujin rhizome(CWR) is a commonly used Chinese herbal medicine for treating blood stasis in China for 1000 of years. However, the underlying mechanism of CWR remains unclear. Aims and Objectives: The purpose of this study is to clarify the bioactive mechanism of CWR in treating blood stasis. Materials and Methods: In this study, pharmacological indexes, including hemorheology and four blood coagulation indexes were tested. Bile and urine metabolomics were engaged by UPLC-Q/TOF-MS. Multivariate statistical analysis were used to screen out differential endogenous metabolites. Results: The results indicated that CWR significantly ameliorated the hemorheology and coagulation functions of acute blood stasis(ABS) model rats. Moreover, 27 endogenous metabolites between the CWR group and the ABS group were screened, and the levels were all improved to certain degrees by CWR preadministration. Metabonomics results indicated that ABS was mainly related to linoleic acid metabolism, arachidonic acid metabolism, glycerophospholipid metabolism, sphingolipid metabolism, pentose and glucuronate intercereasonversions, steroid hormone biosynthesis, and primary bile acid biosynthesis. Conclusion: In a word, the metabolomics method is consistent with the holistic view of traditional Chinese medicine(TCM) that can be a powerful means to illustrate the biological activity mechanism of CWR in treating blood stasis and to offer research demonstration for further study on the effector mechanism of TCM.

微流控芯片液液萃取结合超高效液相色谱-串联质谱法检测玉米油中黄曲霉毒素B1

目的 基于三相层流微流控芯片液液萃取技术在样品前处理上的优势,建立超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)快速检测玉米油中黄曲霉毒素B1的方法。方法 设计了双通道和三通道入口的两种芯片,将含有黄曲霉毒素B1的样品溶液及萃取剂甲醇溶液引入芯片中进行LLE,溶液在芯片通道上分别形成单个和双个稳定的层流界面。将所得的萃取液进行UPLC-MS/MS检测。对样品溶液与萃取剂的流速、芯片萃取通道的宽度等因素进行考察,同时对比两种芯片。结果 样品溶液流速为200μL/h、甲醇流速为300μL/h,萃取通道宽度为200μm的三相层流微流控芯片的萃取效果更好且前处理时间更短。在最优流速下平行实验6次,相对标准偏差(relative standard deviation,RSD)为5.6%,芯片对黄曲霉毒素B1的萃取率为96.8%,检出限为0.05μg/kg,前处理时间为15 min。结论 三相层流微流控芯片比双Y型层流微流控芯片对黄曲霉毒素B1的萃取率更高、萃取时间更短,可为检测其他食品污染物提供参考。

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.

应用液液萃取分析中度烘烤橡木片中挥发性化合物

运用液液萃取与气相色谱-质谱联用技术,研究美国、法国与中国中度烘烤程度橡木片的挥发性化合物。共分离和检测出85种挥发性化合物,包括2种萜烯醇类、1种醛类、7种有机酸类、2种酯类、3种内酯类、7种芳香族类、6种酮类、15种呋喃类、34种酚类,以及8种其他化合物。酚类化合物在橡木中种类较多,浓度较高。定量结果表明,丁香醛、香兰素、糠醛、5-羟甲基糠醛等物质质量浓度较高,对橡木的整体风味影响较大。从呋喃类、橡木内酯、香兰素和丁香醛等化合物比较看,中国橡木与美国橡木类似。

非小细胞肺癌患者尿中Exosomes蛋白质组的差异表达分析

除血浆之外,尿液是另一种寻找潜在生物标志物的重要生物材料。本研究以200000×g超速离心法分离正常人和非小细胞肺癌(NSCLC)患者尿液中的Exosomes,运用1DSDS-PAGE对Exosomes蛋白质组进行分组,从电泳胶上切取正常组和疾病组的20～31kDa条带,胰蛋白酶酶解后,进行HPLC-CHIP-MS/MS分析,并通过UniProtKB/SWISS-PORT数据库搜索鉴定了24种蛋白质,其中在NSCLC患者尿液Exosomes蛋白质组中发现了8种差异表达蛋白,包括免疫球蛋白κ的3个片段、2种Ras相关蛋白、谷胱甘肽S转移酶A2、血清淀粉样P成分前体和磷脂酰乙醇胺结合蛋白1。

液体芯片-飞行时间质谱对结膜松弛症患者泪液蛋白质的分析

目的应用液体芯片-飞行时间质谱系统分析结膜松弛症患者的泪液蛋白质表达谱,寻找潜在具有结膜松弛症诊断意义的生物学标志物。方法收集10例结膜松弛症患者和9例正常对照的泪液,利用毛细管法每眼收集15μL泪液。经ClinProt磁珠纯化、基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析及ClinProTools生物信息学软件研究其泪液蛋白表达谱,比较2组人群泪液蛋白质谱表达的差异。结果 2组共检测发现了77个蛋白质峰,其中13个蛋白质峰差异有统计学意义(P<0.05)。其中质荷比(m/z)为1 632.48和3 401.87者为差异蛋白质,能较好地鉴别结膜松弛症组和正常对照组。结论液体芯片-飞行时间质谱系统可高效、精确地筛查泪液蛋白质。结膜松弛症组与正常对照组泪液蛋白存在差异,值得进一步研究,可能对发现结膜松弛症的发病机制和寻求有效的防治手段有指导作用。

多维色谱串联质谱分析肝癌患者血浆中的低丰度差异蛋白

建立了分析肝癌患者血浆中低丰度差异蛋白的新的多维色谱串联质谱方法。以5例肝细胞癌(HCC)患者血浆为研究对象,选用免疫亲和色谱Agilent MARS Spin Cartridge去除血浆中6种主要高丰度蛋白;离线RP-HPLC预分级血浆低丰度蛋白并收集差异表达蛋白组;液相色谱-芯片(HPLC-CHIP,包含一支纳升级Zorbax300SB-C18富集柱和一支纳升级Zorbax 300SB-C18分析柱)富集、分离差异表达蛋白组胰酶酶解肽混合物,线性离子阱质谱在线分析,以Spectrum Mill MS Proteomics Workbench自动分析MS和MS/MS数据后在UniProtKB/SWISS-PORT数据库中进行搜索,鉴定得到27种差异蛋白,其中23种蛋白与各种疾病或肿瘤相关,IMP3、ARNT2和GRIP1可能成为诊断HCC的潜在生物标志物。

蛋白质组学研究的分离技术及其进展

蛋白质的分离技术是蛋白质组学研究的核心技术 ,目前应用于蛋白质分离的方法主要包括二维电泳分离技术、液相色谱 质谱分离技术以及蛋白质芯片。作者通过文献复习 ,对以上三种技术的基本原理。

基于核酸适配体的微流控芯片酶反应器用于蛋白质的分析

将核酸适配体作为胰蛋白酶固定化介质,制备了一种新型的微流控芯片酶反应器,并与高效液相色谱-串联质谱联用,搭建了在线分析平台;分别使用标准蛋白及混合蛋白样品对芯片的酶解效率及联用平台的分析能力进行了初步评价。结果表明,5 ng肌红蛋白经该平台分析后肽段覆盖率可达到37%;对500 ng混合蛋白进行3次平行分析,肽段覆盖率及相对标准偏差分别为44.3%、6.5%(牛血清白蛋白),65.0%、2.7%(肌红蛋白)和62.0%、5.6%(细胞色素c);初步实验表明,该在线分析平台具有检测灵敏度高、重现性好、酶解效率高的特点,有望在蛋白质组学分析中发挥重要作用。

Using cell membrane chromatography and HPLC-TOF/MS method for in vivo study of active components from roots of Aconitum carmichaeli

An offline two-dimensional system combining a rat cardiac mascle cell membrane chromatography time-of-flight mass spectrometry （CMC-TOF/MS） with a high performance liquid chromatography time-of-flight mass spectrometry （HPLC-TOF/MS） was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis of the analysis of the first dimension, retention components of the urine sample were collected into 30 fractions （one fraction per minute）. Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine （Songorine, 14-benzoylneoline, Deoxyaconitine, etc. ） exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the system suggest that the CMC can be applied to in vivo study.

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.……

Serum metabolic profiling of targeted bile acids reveals potentially novel biomarkers for primary biliary cholangitis and autoimmune hepatitis

BACKGROUND Primary biliary cholangitis(PBC)and autoimmune hepatitis(AIH)are two unexplained immune diseases.The golden standard for diagnosis of these diseases requires a liver biopsy.Liver biopsy is not widely accepted by patients because of its invasive nature,and atypical liver histology can confuse diagnosis.In view of the lack of effective diagnostic markers for PBC and AIH,combined with the increasingly mature metabolomics technologies,including full-contour metabolomics and target.AIM To determine non-invasive,reliable,and sensitive biochemical markers for the differential diagnosis of PBC and AIH.METHODS Serum samples from 54 patients with PBC,26 patients with AIH and 30 healthy controls were analyzed by Ultra-high performance liquid chromatographytandem mass spectrometry serum metabolomics.The metabolites and metabolic pathways were identified,and the metabolic changes,metabolic pathways and inter-group differences between PBC and AIH were analyzed.Fifteen kinds of target metabolites of bile acids(BAs)were quantitatively analyzed by SRM,and the differential metabolites related to the diagnosis of PBC were screened by receiver operating characteristic curve analysis.RESULTS We found the changes in the levels of amino acids,BAs,organic acids,phospholipids,choline,sugar,and sugar alcohols in patients with PBC and AIH.Furthermore,the SRM assay of BAs revealed the increased levels of chenodeoxycholic acid,lithocholic acid(LCA),taurolithocholic acid(TLCA),and LCA+TLCA in the PBC group compared with those in the AIH group.The levels of BAs may be used as biomarkers to differentiate PBC from AIH diseases.The levels of glycochenodeoxycholic acid,glycochenodeoxycholic sulfate,and taurodeoxycholic acid were gradually elevated with the increase of Child-Pugh class,which was correlated with the severity of disease.CONCLUSION The results demonstrated that the levels of BAs could serve as potential biomarkers for the early diagnosis and assessment of the severity of PBC and AIH.

亲水作用高效液相色谱-串联质谱法测定薯片及饼干中丙烯酰胺的含量

目的建立亲水作用高效液相色谱-串联质谱法测定薯片及饼干中丙烯酰胺含量的分析方法。方法薯片及饼干经超纯水提取,正己烷除脂,Cleanert■ACA固相萃取柱净化,BEH Amide色谱柱(2.1mm×100mm,1.7μm)分离,并以0.1%(V:V)甲酸水-乙腈溶液=2:8(V:V)为流动相,电喷雾正离子模式电离,多反应监测模式检测,内标法定量。结果丙烯酰胺在5~2000μg/L范围内线性关系良好,相关系数大于0.999。方法的检出限(S/N>3)和定量限(S/N>10)分别为3μg/kg和10μg/kg。在不同基质中,丙烯酰胺在20、200、1000μg/kg的添加水平下平均加标回收率为91.5%~98.2%,相对标准偏差为2.71%~4.82%。结论此方法具有准确度好、灵敏度高等特点,适用于薯片及饼干中丙烯酰胺的测定。

UPLC-QTOF/MS Method for Screening and Determination of 59 Non-labeled Components in Veterinary Drug Preparations

[Objectives]This study was conducted to provide an accurate and reliable method for rapid high-throughput screening of veterinary drug preparations.[Methods]For the matrixes of veterinary drug preparations,high-performance liquid chromatography-high resolution quadrupole time-of-flight mass spectrometry(UPLC-QTOF/MS)was used to establish a fast screening method for 59 non-standard components in five categories of antiviral agents,aminoglycosides,quinolones,sulfonamides,and tetracyclines in veterinary drugs.The target drugs were separated by a Waters ACQUITY UPLC HSS T3 chromatographic column(50 mm×2.1 mm,1.8μm),and data were collected in the positive ion mode.Good separation of the 59 drugs was achieved within 7 min.[Results]In the concentration range of 0-100 ng/ml,each drug showed a good linear relationship,and the correlation coefficients were all greater than 0.999.The detection limits of the 59 drugs were in the range of 0.1-0.5 mg/ml,and the recovery under the addition concentration of 5 mg/ml was in the range of 85.2%-103.8%.[Conclusions]The method is fast,simple,accurate,and highly sensitive,and is suitable for high-throughput screening and qualitative identification of non-standard components in veterinary drug preparations.

芯片液相色谱-质谱法高灵敏度检测水产品中微囊藻毒素

建立了一种芯片液相色谱-高分辨质谱测定3种微囊藻毒素(MC-RR、MC-YR和MC-LR)的分析方法.在最优条件下,3种藻毒素在16min内实现基线分离,并选择电喷雾源正离子模式进行质谱测定.方法在2.5~2000ng/mL范围内线性良好,线性相关系数大于0.9965,检测限介于0.5~2.0ng/mL之间.鱼肉和虾肉样品经固相萃取法净化处理后未测得上述毒素残留,加标回收率为82.8%~120.3%.方法具有样品用量少、定性快速准确、灵敏度高等优点,适合用于水产品等生物样品中藻毒素的痕量监测.