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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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%.

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.

Study on the Structural Effect of Maltoligosaccharides on Cytochrome c Complexes Stabilities by Native Mass Spectrometry

Noncovalent interactions between ligands and targeting proteins are essential for understanding molecular mechanisms of proteins.In this work,we investigated the interaction of Cytochrome c(Cyt c)with maltoligosaccharides,namely maltose(Mal Ⅱ),maltotriose(Mal Ⅲ),maltotetraose(Mal Ⅳ),maltopentaose(Mal Ⅴ),maltohexaose(Mal Ⅵ)and maltoheptaose(Mal Ⅶ).Using electrospray ionization mass spetrometry(ESI-MS)assay,the 1:1 and 1:2 complexes formed by Cyt c with maltoligosaccharide ligand were observed.The corresponding association constants were calculated according to the deconvoluted spectra.The order of the relative binding affinities of the selected oligosaccharides with Cyt c were as MalⅢ>MalⅣ>MalⅡ>MalⅤ>MalⅥ>MalⅦ.The results indicated that the stability of noncovalent protein complexes was intimately correlated to the molecular structure of bound ligand.The relevant functional groups that could form H-bonds,electrostatic or hydrophobic forces with protein’s amino residues played an important role for the stability of protein complexes.In addition,the steric structure of ligand was also critical for an appropriate interaction with the binding pocket of proteins.

Nanokit coupled electrospray ionization mass spectrometry for analysis of angiotensin converting enzyme 2 activity in single living cell

Angiotensin-converting enzyme 2(ACE2) is not only an enzyme but also a functional receptor on cell membrane for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). Here, the activity of ACE2 in single living cell is firstly determined using a nanokit coupled electrospray ionization mass spectrometry(nanokit-ESI-MS). Upon the insertion of a micro-capillary into the living h ACE2-CHO cell and the electrochemical sorting of the cytosol, the target ACE2 enzyme hydrolyses angiotensin II inside the capillary to generate angiotensin 1-7. After the electrospray of the mixture at the tip of the capillary, the product is differentiated from the substrate in molecular weight to achieve the detection of ACE2 activity in single cells. The further measurement illustrates that the inflammatory state of cells does not lead to the significant change of ACE2 catalytic activity, which elucidates the relationship between intracellular ACE2 activity and inflammation at single cell level. The established strategy will provide a specific analytical method for further studying the role of ACE2 in the process of virus infection, and extend the application of nanokit based single cell analysis.

磁性氮化碳复合材料的制备及其对磷酸化肽的富集

蛋白质的磷酸化在细胞信号传导和疾病发生发展中起着重要作用,但磷酸化的动态变化和低丰度的特点使得对其直接分析有着较大的困难。为了解决磷酸化肽难离子化、检测丰度低的瓶颈问题,本研究制备了一种磁性氮化碳复合材料,结合基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),建立了一种对复杂样品中低丰度磷酸化肽富集与分析的方法。采用电子显微镜、红外光谱分析及X射线衍射分析等手段对合成的磁性氮化碳材料进行表征。以酪蛋白酶解产物为实验模型,发现磁性氮化碳材料能够实现对磷酸化肽的高选择性富集和高灵敏度检测,检出限为0.1 fmol。选择脱脂牛奶、人唾液和人血清为实际分析样品,发现磁性氮化碳材料对微量蛋白生物样品中磷酸化肽的分析具有较高的应用潜力。

Real-time toxicity prediction of Aconitum stewing system using extractive electrospray ionization mass spectrometry

Due to numerous obstacles such as complex matrices,real-time monitoring of complex reaction systems(e.g.,medicinal herb stewing system)has always been a challenge though great values for safe and rational use of drugs.Herein,facilitated by the potential ability on the tolerance of complex matrices of extractive electrospray ionization mass spectrometry,a device was established to realize continuous sampling and real-time quantitative analysis of herb stewing system for the first time.A complete analytical strategy,including data acquisition,data mining,and data evaluation was proposed and implemented with overcoming the usual difficulties in real-time mass spectrometry quantification.The complex Fuzi(the lateral root of Aconitum)-meat stewing systems were real-timely monitored in150 min by qualitative and quantitative analysis of the nine key alkaloids accurately.The results showed that the strategy worked perfectly and the toxicity of the systems were evaluated and predicated accordingly.Stewing with trotters effectively accelerated the detoxification of Fuzi soup and reduced the overall toxicity to 68%,which was recommended to be used practically for treating rheumatic arthritis and enhancing immunity.The established strategy was versatile,simple,and accurate,which would have a wide application prospect in real-time analysis and evaluation of various complex reaction systems.

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。

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。