High Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometric Analysis of Bilobalide and Ginkgolides in Ginkgo biloba L. Leaves

The ginkgo terpenoids including bilobalide and ginkgolides are the main pharmaceutical components in the leaves or extracts of Ginkgo biloba L. In this paper, the analysis of bilobalide and ginkgolides in leaves of Ginkgo biloba L. by high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry (MS) was carried out. The separation was performed on Inertsil ODS3 column with methanol-water (36:64) as mobile phase, with 1 mL·min -1 of flow rate at 35℃. Then the mass spectrum analysis was conducted by ZMD micromass electrospray ionization (ESI)-mass spectrometer (MS). The HPLC total ion chromatogram and selected ion chromatogram (with 325, 407, 423, 439 of m/z) of the sample and ESI-/MS mass spectra of the peaks in the chromatograms were obtained. So bilobalide, ginkgolide A, B, C and J in Ginkgo biloba L. leaves were identified. The method is easy and rapid, with a good accuracy.

Identification,structure elucidation and origin of a common pyridinium-thiocyanate intermediate in electrospray mass spectrometry among the benziamidazole-class proton pump inhibitors

During the analysis of benziamidazole-class irreversible proton pump inhibitors,an unusual mass spectral response with the mass-to-charge ratio at[Mt10]t intrigued us,as it couldn't be assigned to any literature known relevant structure,intermediate or adduct ion.Moreover,this mysterious mass pattern of[Mt10]t has been gradually observed by series of marketed proton pump inhibitors,viz.omeprazole,pantoprazole,lansoprazole and rabeprazole.All the previous attempts to isolate the corresponding component were unsuccessful.The investigation of present work addresses this kind of signal to a pyridinium thiocyanate mass spectral intermediate(10),which is the common fragment ion of series of labile aggregates.The origin of such aggregates can be traced to the reactive intermediates formed by acid-promoted degradation.These reactive intermediates tend to react with each other and give raise series of complicated aggregates systematically in a water/acetonitrile solution by electrospray ionization.The structure of the corresponding pyridinium thiocyanate species of omeprazole(10a)has been eventually characterized with the help of synthetic specimen(10a′).Our structural proposal as well as its origin was supported by in situ nuclear magnetic resonance,chemical derivatization and colorimetric experiments.

Rapid authentication of different herbal medicines by heating online extraction electrospray ionization mass spectrometry

The rapid and accurate authentication of traditional Chinese medicines(TCMs)has always been a key scientific and technical problem in the field of pharmaceutical analysis.Herein,a novel heating online extraction electrospray ionization mass spectrometry(H-oEESI-MS)was developed for the rapid and direct analysis of extremely complex substances without the requirement for any sample pretreatment or pre-separation steps.The overall molecular profile and fragment structure features of various herbal medicines could be completely captured within 10–15 s,with minimal sample(<0.5 mg)and solvent consumption(<20μL for one sample).Furthermore,a rapid differentiation and authentication strategy for TCMs based on H-oEESI-MS was proposed,including metabolic profile characterization,characteristic marker screening and identification,and multivariate statistical analysis model validation.In an analysis of 52 batches of seven types of Aconitum medicinal materials,20 and 21 key compounds were screened out as the characteristic markers of raw and processed Aconitum herbal medicines,respectively,and the possible structures of all the characteristic markers were comprehensively identified based on Compound Discoverer databases.Finally,multivariate statistical analysis showed that all the different types of herbal medicines were well differentiated and identified(R^(2)X>0.87,R^(2)Y>0.91,and Q^(2)>0.72),which further verified the feasibility and reliability of this comprehensive strategy for the rapid authentication of different TCMs based on H-oEESI-MS.In summary,this rapid authentication strategy realized the ultra-high-throughput,low-cost,and standardized detection of various complex TCMs for the first time,thereby demonstrating wide applicability and value for the development of quality standards for TCMs.

Studies on the Basic Conformation of Lysozyme by Electrospray Ionization-Mass Spectrometry(ESI-MS)

In the present work, 2-mercaptoethanol and dithiothreitol were used to hydrogenise the internal disulfide bond in lysozyme. The experimental results indicate that the charge distribution of the proteins are different in the reaction process. From the calculated molecular weight, the reduction process of the disulfide bond in the molecules can be described, and the number of the disulfide bonds in the molecule can also be determined.

Analysis of Norditerpenoid Alkaloids Extracted from Aconitum sinomantanum Nakai by Electrospray Ionization Tandem Mass Spectrometry

Electrospray ionization mass spectrometry（ESI-MS） was applied simultaneously in determining norditerpenoid alkaloids from the roots of Aconitum sinomantanum Nakai （RAS） based on molecular mass information. The tandem mass spectra（ ESI-MS^n） provided the alkaloidal structural information, through which the existence of these alkaloids was further confirmed. Accordingly, six known norditerpenoid alkaloids were simultaneously determined on the basis of their ESI-MS^n spectra. Furthermore, based on the diagnostic fragmentation pathways of alkaloidal MS^n, a rapid method for direct detection and characterization of alkaloids from an ethanolic extract of RAS was described.

Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry for Simultaneous Determination of Metformin and Glimepiride in Beagle Dog Plasma and Bioequivalence Study

A sensitive and selective liquid chromatography-electrospray ionization tandem mass spectrometry（LC- ES1-MS/MS） was used for the simultaneous determination of metformin and glimepiride in beagle dog plasma with glipizide as internal standard（IS）. After simplified protein precipitation with methanol, both the analytes and IS were chromatographed on a Zorbax CN column via gradient elution with methanol（containing 5 mmol/L ammonium ace- tate） and 5 mmol/L aqueous ammonium acetate as the mobile phase. Detection was performed by multiple reaction monitoring（MRM） scanning via ESI source operated in positive ionization mode. Specificity, linearity, accuracy, pre- cision, recovery, matrix effect and stability were validated for metformin and glimepiride in beagle dog plasma. The calibration curves were linear in a concentration range of 10-10000 ng/mL for metformin and 4-4000 ng/mL for glimepiride with both correlation coefficients higher than 0.99. The recoveries obtained for the analytes and IS were all between 82.7% and 101.2%. The method exhibited excellent performance in terms of selectivity, robustness, short analytical time and simplicity of sample preparation. Finally, the proposed method was applied to a bioequivalence study of self-made bilayer tablet and commercial formulation containing 500 mg of metformin and 1 mg of glimepi- ride in beagle dogs.

Fast Screening of Chicken Egg Lysozyme in White Wine Products by Extractive Electrospray Ionization Mass Spectrometry

Fast detection of trace lysozyme,one of the most important food allergens in white wine samples,was achieved by extractive electrospray ionization mass spectrometry without sample pretreatment in this study.The multiply-charged ions of m/z1587 were chosen for the quantitative detection of lysozyme in white wine,showing linear dynamic signal responses in a range of 5―75μg/mL with a linearity coefficient of 0.999 and an acceptable relative standard deviation（RSD） of 8.0%―15.0% for directly measuring lysozyme in the complex food samples.The limit of detection for lysozyme in white wine sample was calculated to be 5 μg/mL,which was lower than the amounts that can provoke allergic reactions（oral test with 3 mg or labial test with 1 mg/mL）.A single sample analysis was completed within 1 min.The data demonstrate that extractive electrospray ionization mass spectrometry is a useful tool for fast screening lysozyme in the complex matrix,showing promising application in the rapid detection of food allergen.

Studies on Fragmentation Pathways of N-Ethoxy(phenyl) phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

The positive and negative ESI-MS/MS spectra of N-ethoxy（phenyl） phosphoryl amino acids（EPP-AA） were investigated by electrospray ionization（ESI） ion trap mass spectrometry. The fragmentation pathways of [ M ＋ Na]^＋ and [ M - H]^- ions are proposed and rationalized. The observation may have some potential applications in the interpretation of the MS/MS spectra of novel N-phosphoryl compounds. The complexity of MS/MS spectra of EPP-AA [ M ＋ Na]^＋ ions is decreased compared with that of N-dialkyloxyphosphoryl amino acid. Therefore, the new phosphonamidate method may be considered one of the superior methods that can be used in sequencing peptides and proteins extensively.

The Application of Electrospray Tandem Mass Spectrometry for <i>de novo</i>Sequencing of Reduced Insulins

Insulin, a blood glucose level mediator, is the mainstream therapeutic choice for diabetes patients. Since patent protection for many originator products is about to expire, manufacturers of follow-on insulin are determined to get their products authorized. According to regulations, a fundamental requirement for biosimilar compounds is that the chemical structure should be the same as that of the originator drug. Hence, the application of qualitative analysis for insulin products is essential during the production and development of biosimilars. In this study, the electrospray tandem MS/MS based de novo sequencing method was developed and validated by analyzing two insulin products with similar primary structures, namely recombinant human insulin and insulin aspart. The results indicated that the complete sequences of both reduced insulins are largely identifiable, although differentiation between leucine and isoleucine is not achieved. More importantly, the observed mass accuracy was substantial. The method can, therefore be applied to quality control and drug development.

Unraveling enhanced membrane lipid biosynthesis in Chlamydomonas reinhardtii starchless mutant sta6 by using an electrospray ionization mass spectrometry-based lipidomics method

The unicellular green alga Chlamydomonas reinhardtii,a well-established model organism,has been widely used in dissecting glycerolipid metabolism in oxygenating photosynthetic organisms.In previous studies,it has been found that shunting carbon precursors from the starch synthesis pathway can lead to a 10-fold increase in TAG content as compared to the wild type,but it is unknown whether inactivation of AGPase may affect membrane lipids biosynthesis.The study aims to investigate global changes in lipid metabolism and homeostasis in the starchless mutant C.reinhardtii sta6.By utilizing an electrospray ionization/mass spectrometry(ESI/MS)-based lipidomics approach,a total of 105 membrane lipid molecules of C.reinhardtii were resolved,including 16 monogalactosyldiacylglycerol(MGDG),16 digalactosyldiacylglycerol(DGDG),11 phosphatidylglycerol(PG),6 sulfoquinovosyldiacylglycerol(SQDG),49 diacylglyceryl-N,N,N-trimethylhomoserine(DGTS),2 phosphatidylethanolamine(PE),and 5 phosphatidylinositol(PI)molecules.The quantitative results indicated that the membrane lipid profiles were similar between the two C.reinhardtii strains grown under both low-and high-light conditions,but the cellular contents of a great number of lipids were altered in sta6 due to the defect in starch biosynthesis.Under low-light conditions,sta6 accumulated more PI,MGDG,DGDG but less amounts of DGTS as compared to WT.Under high light,sta6 cells contained higher content membrane lipids than cc-124,except for PG,which is more or less similar in both strains.Our results demonstrate that the cellular membrane lipid homeostasis underwent profound changes in the starchless mutant,and thereby its physiological impact remains to be explored.

Effects of Temperature and Energy on Stability of Oligomeric Enzyme Probed on Electrospray Ionization Mass Spectrometry

Escherichia coli 3-Deoxy-D-manno-octulosonate 8-phosphate（KDO8P） synthase catalyzed the condensation reaction between D-arabinose 5-phosphate（A5P） and phosphoenolpyruvate（PEP） to form KDO8P and inorganic phosphate（Pi）. The noncovalent tetrameric association ofKDO8P synthase was observed and dissociated in gas phase by means of electrospray ionization mass spectrometry under the very ＂soft＂ conditions. The results indicate that PEP-bound enzyme generated abundant tetrameric species as well as monomeric species at the ＂soft＂ conditions, whereas, the unbound enzyme favored the formation of a dimeric species. The mass spectra of the mixture of the enzyme with one of substrates, PEP, and A5P or one of products, KDO8P and Pi show that the complex of the unbound enzyme with PEP or Pi was prone to the formation of a monomeric species, whereas, that of the unbound enzyme with A5P or KDO8P was similar to the unbound enzyme. The intensity of the dimeric species increased with the increase of temperature at a collision voltage of 10 V. Taken together, the results presented here suggest that mass spectrometry will be a powerful tool to explore subtile conformational changes and/or subunit-subunit interactions of multiprotein assembly induced by ligand-binding and/or the changes of environmental conditions.

Molecular Probes in Tandem Electrospray Ionization Mass Spectrometry: Application to Tracing Chemical Changes of Specific Phospholipid Molecular Species

New ionization and detection techniques in mass spectrometry have been successfully applied for efficient analyses of complex biological systems. It is, however, still difficult to trace structural changes of a specific molecular species in such systems. In the present study, a molecular probe strategy in combination with tandem electrospray ionization mass spectrometry has been examined using synthetic deuterium-labeled phosphatidylcholine hydroperoxide (PC-OOH/D3) and ethyl-labeled phosphatidylcholine having docosahexaenoic acid side chain (DHA-PC/Et). Administration of a mixture of PC-OOH/D3 and DHA-PC/Et to human blood and human skin surface, followed by extraction and analysis with collision-induced tandem electrospray ionization mass spectrometry demonstrated that metabolites of both molecular probes can be detected simultaneously with strict selectivity. The present method is also found to be useful in tracing chemical changes of the unstable docosahexaenoyl group on the surface of processed fish. The activity of phospholipase A2 can also be assessed using a phospholipid molecular probe with a linoleoyl and a deuteriomethyl group via selective detection of the lyso-phospholipid product by mass spectrometry. The advantage of the present method is that no chromatographic separation is required and analysis can be performed under strictly the same condition for different molecular probes, affording multiple data by one experiment. The present strategy may be useful for tracing time-dependent phenomena in dynamic phospholipid biochemistry, and can be widely used for any biological and food systems.

Comparison of different approaches for direct coupling of solid-phase microextraction to mass spectrometry for drugs of abuse analysis in plasma

The direct coupling of solid-phase microextraction(SPME)to mass spectrometry(MS)(SPME-MS)has proven to be an effective method for the fast screening and quantitative analysis of compounds in complex matrices such as blood and plasma.In recent years,our lab has developed three novel SPME-MS techniques:SPME-microfluidic open interface-MS(SPME-MOI-MS),coated blade spray-MS(CBS-MS),and SPME-probe electrospray ionization-MS(SPME-PESI-MS).The fast and high-throughput nature of these SPME-MS technologies makes them attractive options for point-of-care analysis and anti-doping testing.However,all these three techniques utilize different SPME geometries and were tested with different MS instruments.Lack of comparative data makes it difficult to determine which of these methodologies is the best option for any given application.This work fills this gap by making a comprehensive comparison of these three technologies with different SPME devices including SPME fibers,CBS blades,and SPME-PESI probes and SPME-liquid chromatography-MS(SPME-LC-MS)for the analysis of drugs of abuse using the same MS instrument.Furthermore,for the first time,we developed different desorption chambers for MOI-MS for coupling with SPME fibers,CBS blades,and SPME-PESI probes,thus illustrating the universality of this approach.In total,eight analytical methods were developed,with the experimental data showing that all the SPME-based methods provided good analytical performance with R^(2)of linearities larger than 0.9925,accuracies between 81%and 118%,and good precision with an RSD%≤13%.

Studies on Fragmentation of Peptide by Nano-electrospray Ionization Fourier Transform Ion Cyclotron Resonance Multi-stage Tandem Mass Spectrometry

The sequence analysis of peptides was performed by nano-electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry（Nano-ESI-FT-ICR-MSn） and several peptides were chosen as examples. With the aid of the collision induced dissociation（CID）, FT-ICR provides not only precise mass/charge ratio, but also structure information of the selected peptides. The fragment ions were identified according to the observed molecular weights and peptide sequence was determined successfully. So Nano-ESI-FT-ICR-MSn is a useful tool for identification of the amino acid sequence of peptides with high confidence. Besides, a pathway for the dehydration of y ions without amino acids containing carboxylic acid under sustained off-resonance irradiation collision-induced dissociation（SORI-CID） condition was proposed.

一例三(5-氯苯并噻唑-2-甲基)胺Fe(Ⅱ)配合物的串联合成过程及电催化性质研究

三(芳基甲基)胺由于强配位性,在氧气活化的单/双金属非血红素金属蛋白类似物的合成中起着重要作用,且能够作为加速配体催化Click反应,因而受到广泛关注。本研究以苯并[d]噻唑-2-基甲胺(S1)和5-氯苯并[d]噻唑-2-基)甲醇(L1)为原料,在FeCl_(3)·6H_(2)O的催化下,串联合成得到了一例新颖的三(5-氯苯并噻唑甲基)胺Fe(Ⅱ)配合物1。用X-射线单晶衍射仪、X-射线粉末衍射仪、电喷雾离子化质谱确证了结构,通过晶体学和电喷雾离子质谱相结合,研究了反应过程,检测到了一系列关键中间体片段([i-Cl]^(+)、[ii+H]^(+)、[iii-Cl]^(+)),结合反应过程跟踪提出了可能的反应机理,包括N-烷基化,氧化水解等步骤,实现了3个共价键的连续构筑。对配合物1进行了OER(析氧反应)测试及稳定性性能研究,其中过电势为391 mV,Tafel斜率为95.9 mV·dec^(-1),在电极密度10 mA/cm^(2)下进行了10 h的恒电流电解,在该时间范围内稳定性良好。通过对配合物1的电催化性能研究则为后续该配体的应用领域提供一种新的方向。

Simultaneous determination of seven flavonoids in Epimedium by liquid chromatography-tandem mass spectrometry method

In this paper, a sensitive and specific liquid chromatography-electrospray ionisation-mass spectrometry （LC-ESI-MS） method has been developed and validated for the identification and determination of seven flavonoids, namely epimedin A, epimedin B, epimedin C, icariin, sagittatoside B, 2＂-O-rhamnosyl icariside II, and baohuoside I in Epimedium from different sources.

基于质谱的单细胞分析技术研究新进展

细胞是生物体最基本的结构和功能单位。近年来,人们对细胞异质性的关注越来越多。为了分析细胞的这种异质性,避免单细胞信息被群体细胞的平均值所淹没,单细胞分析技术不断发展起来。由于单细胞的体积小,内含物质含量低且种类繁多,某些物质在细胞内的变化迅速,分析过程中不同物质之间相互干扰,造成了单细胞分析极具挑战性。基于质谱的单细胞分析技术具有通用性强、无需标记、高灵敏度、高分辨率、高选择性的优点,已逐渐在单细胞分析技术中脱颖而出,成为单细胞分析的理想工具。本文系统地总结了近5年来基于质谱的单细胞分析技术的最新进展,包括电喷雾电离质谱、激光解吸电离质谱、二次离子质谱、电感耦合等离子体质谱,并对未来的研究和技术方向进行展望,希望为单细胞质谱分析技术的研究提供参考。

探针电喷雾离子化质谱法结合人工智能建立肺腺癌术中良恶性快速诊断模型

目的:采用探针电喷雾离子化质谱技术对术中送检肺标本进行分析,结合人工智能技术建立一种快速良恶性诊断模型并对其进行评价。方法:收集中日友好医院病理科2022年7—12月送检的肺腺癌手术切除标本47例,以及周边非癌组织。经称量、提取、离心等处理,将上清液直接用快速原位离子化质谱仪DPiMS QT进行扫描分析。使用Anaconda软件自主研发程序进行数据读取、预处理、多变量分析、特征筛选、模型训练和优化。采用Metaboanalyst■对肺腺癌中发现的特征代谢物进行了代谢通路分析。结果:PLS regression方法降维分析结果,将199个特征降维成10个主成分用于区分癌与非癌组织。使用支持向量机(SVM)、随机森林(RF)和多层感知器(MLP)等10种算法进行建模后发现,基于MLP建立的分类模型的交叉验证准确率最好,判定的20重交叉验证准确率达99%±2%。代谢通路分析发现肺腺癌中谷氨酰胺下调、谷氨酸上调,其变化规律符合肺癌中通过谷氨酸-TCA循环促进蛋白质合成的规律。结论:应用探针电喷雾离子化质谱法结合人工智能技术可以在几分钟内将肺部良恶性病变区分开,实现肺组织新鲜标本的快速、准确分类,辅助肺组织的术中快速诊断。此外,肺腺癌中差异表达的物质还可以用于进一步的肿瘤代谢研究。

无鞘流毛细管电泳-电喷雾串联质谱用于面粉中荧光增白剂的高灵敏检测

食品中残留的过量荧光增白剂(FWAs)对人体健康存在潜在威胁,因此,开发准确、高灵敏的FWAs检测方法在食品安全监测方面具有重要意义。本工作提出了利用无鞘流接口的毛细管电泳-电喷雾串联质谱(CE-ESI-MS/MS)的方法,以实现面粉样品中6种FWAs的高灵敏检测。实验采用超声辅助液相萃取法进行样品前处理,以减小复杂样品中基质效应的干扰。以氯仿-甲醇(3∶2,v/v)溶液作为萃取剂,在30℃下对样品中的FWAs进行萃取。萃取完成后,经离心、氮气吹干后用氯仿-甲醇(1∶4,v/v)复溶后检测。无鞘流CE-ESI-MS/MS方法采用正离子(ESI+)和多反应监测(MRM)模式,利用二级质谱对6种目标物同时进行定性定量分析,从而提高方法的检测通量和灵敏度。结果显示,本方法具有较宽的线性范围、良好的线性关系和较低的方法检出限(0.04~0.67 ng/g),在实际样品中3个水平下的加标回收率良好(86.2%~103.7%),日间和日内重复性(RSD)分别不大于11.5%和10.2%。上述研究表明,本方法适用于复杂基质中多种FWAs的准确、高灵敏检测,在面粉样品的质量评估和FWAs的污染监控方面具有潜在的应用价值。

质谱离子源内裂解和重排可能会为化学反应中间体的捕获带来错误信息

质谱法广泛应用于在线反应监测和高活性中间体捕获,极大助力了化学反应机理研究。微液滴具有极端的化学环境,有望捕获高活性中间体,与质谱联用可实现原位表征。有报道表明,微液滴表面的局部强酸环境可以稳定短寿命的碳正离子,结合在线质谱可以检测其信号。本文使用电喷雾电离质谱(ESI-MS)测定了一系列碳正离子的前体分子,在排除微液滴效应影响的前提下,检测到这些碳正离子信号。进一步的实验表明,受进样毛细管高温和管透镜电压的共同影响,在质谱的粗真空区域,前体物种会发生源内裂解产生碳正离子碎片,这些碎片可能会被误判为液滴反应中产生的高活性中间体,从而影响反应机理研究。本工作表明,在使用质谱进行反应监测和中间体捕获研究时,应谨慎地考察各仪器参数,尤其是离子光学系统所加电压对离子的影响,以避免对化学反应机制的误判。