中药质谱分析方法及应用研究

分别从中药成分分析、活性筛选和代谢组学三方面对质谱技术在中药研究中的应用进展进行了全面综述.在中药成分分析方面,重点介绍了寡糖异构体的分析方法,以及质谱指纹图谱技术在中药成分分析及质量控制中的应用;在活性筛选方面,分别介绍了超滤-质谱、细胞膜色谱-质谱、微透析-质谱、亲和色谱-质谱、强度衰减质谱、修饰琼脂糖珠-质谱和直接分析质谱等技术及其应用;在代谢组学研究方面,对中药治疗肝损伤、肾虚、心肌梗死和糖尿病等疾病方面的研究进展进行了阐述.上述内容充分反映了质谱技术在中药创新性研究中的重要性.     

Characterization of tetrathiofulvalene compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Tetrathiofulvalene compounds are important components of charge-transfer complexes, which may be applied in various fields of scientific research and practical applications. Some of these compounds cannot be characterized by mass spectrometry. Here, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for the characterization of tetrathiofulvalenes. The samples could be easily desorbed and ionized to form singly charged ions, and mass spectra with isotopic resolution readily obtained. The mass spectrometric results for 26 compounds have shown that MALDI-TOF is more effective and convenient than other mass spectrometry methods, and resolves the problem of mass spectrometric characterization of tetrathiofulvalene compounds.     

Biomedical and biological mass spectrometry.

This review focuses on biological and biomedical mass spectrometry, and covers a selection of publications in this area included in the MEDLINE database for the period 1987-2001. Over the last 15 years, biological and biomedical mass spectrometry has progressed out of all recognition. The development of soft ionization methods, such as electrospray ionization and matrix-assisted laser desorption ionization, has mainly contributed to the remarkable progress, because they can easily produce gas-phase ions of large, polar, and thermally labile biomolecules, such as proteins, peptides, nucleic acids and others. The innovations of ionization methods have led to remarkable progress in mass spectrometric technology and in biochemistry, biotechnology and molecular biology research. In addition, mass spectrometry is one of the powerful and effective technologies for drug discovery and development. It is applicable to studies on structural determination, drug metabolism, including pharmacokinetics and toxicokinetics, and de novo drug discovery by applying post-genomic approarches. In the present review, the innovative soft ionization methods are first discussed along with their features. Also, the characteristics of the mass spectrometers which are active in the biological and biomedical research fields are also described. In addition, examples of the applications of biological and biomedical mass spectrometry are provided.     

"Affinity-proteomics": direct protein identification from biological material using mass spectrometric epitope mapping

We describe here a new approach for the identification of affinity-bound proteins by proteolytic generation and mass spectrometric analysis of their antibody bound epitope peptides (epitope excision). The cardiac muscle protein troponin T was chosen as a protein antigen because of its diagnostic importance in myocardial infarct, and its previously characterised epitope structure. Two monoclonal antibodies (IgG1-1B10 and IgG1-11.7) raised against intact human troponin T were found to be completely cross reactive with bovine heart troponin T. A combination of immuno-affinity isolation, partial proteolytic degradation (epitope excision), mass spectrometric peptide mapping, and database analysis was used for the direct identification of Tn T from bovine heart cell lysate. Selective binding of the protein was achieved by addition of bovine heart cell lysate to the Sepharose-immobilised monoclonal antibodies, followed by removal of supernatant material containing unbound protein. While still bound to the affinity matrix the protein was partially degraded thereby generating a set of affinity-bound, overlapping peptide fragments comprising the epitope. Following dissociation from the antibody the epitope peptides were analysed by matrix assisted laser desorption-ionisation (MALDI) and electrospray-ionisation (ESI) mass spectrometry. The peptide masses identified by mass spectrometry were used to perform an automated database search, combined with a search for a common "epitope motif". This procedure resulted in the unequivocal identification of the protein from biological material with only a minimum number of peptide masses, and requiring only limited mass-determination accuracy. The dramatic increase of selectivity for identification of the protein by combining the antigen-antibody specificity with the redundancy of peptide sequences renders this "affinity-proteomics" approach a powerful tool for mass spectrometric identification of proteins from biological material.     

Inorganic trace analysis by mass spectrometry

Mass spectrometric methods for the trace analysis of inorganic materials with their ability to provide a very sensitive multielemental analysis have been established for the determination of trace and ultratrace elements in high-purity materials (metals, semiconductors and insulators), in different technical samples (e.g. alloys, pure chemicals, ceramics, thin films, ion-implanted semiconductors), in environmental samples (waters, soils, biological and medical materials) and geological samples. Whereas such techniques as spark source mass spectrometry (SSMS), laser ionization mass spectrometry (LIMS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), glow discharge mass spectrometry (GDMS), secondary ion mass spectrometry (SIMS) and inductively coupled plasma mass spectrometry (ICP-MS) have multielemental capability, other methods such as thermal ionization mass spectrometry (TIMS), accelerator mass spectrometry (AMS) and resonance ionization mass spectrometry (RIMS) have been used for sensitive mono- or oligoelemental ultratrace analysis (and precise determination of isotopic ratios) in solid samples. The limits of detection for chemical elements using these mass spectrometric techniques are in the low ng g⁻¹ concentration range. The quantification of the analytical results of mass spectrometric methods is sometimes difficult due to a lack of matrix-fitted multielement standard reference materials (SRMs) for many solid samples. Therefore, owing to the simple quantification procedure of the aqueous solution, inductively coupled plasma mass spectrometry (ICP-MS) is being increasingly used for the characterization of solid samples after sample dissolution. ICP-MS is often combined with special sample introduction equipment (e.g. flow injection, hydride generation, high performance liquid chromatography (HPLC) or electrothermal vaporization) or an off-line matrix separation and enrichment of trace impurities (especially for characterization of high-purity materials and environmental samples) is used in order to improve the detection limits of trace elements. Furthermore, the determination of chemical elements in the trace and ultratrace concentration range is often difficult and can be disturbed through mass interferences of analyte ions by molecular ions at the same nominal mass. By applying double-focusing sector field mass spectrometry at the required mass resolution—by the mass spectrometric separation of molecular ions from the analyte ions—it is often possible to overcome these interference problems. Commercial instrumental equipment, the capability (detection limits, accuracy, precision) and the analytical application fields of mass spectrometric methods for the determination of trace and ultratrace elements and for surface analysis are discussed.     

Trends in single-cell analysis by use of ICP-MS

The analysis of single cells is a growing research field in many disciplines such as toxicology, medical diagnosis, drug and cancer research or metallomics, and different methods based on microscopic, mass spectrometric, and spectroscopic techniques are under investigation. This review focuses on the most recent trends in which inductively coupled plasma mass spectrometry (ICP-MS) and ICP optical emission spectrometry (ICP-OES) are applied for single-cell analysis using metal atoms being intrinsically present in cells, taken up by cells (e.g., nanoparticles), or which are artificially bound to a cell. For the latter, especially element tagged antibodies are of high interest and are discussed in the review. The application of different sample introduction systems for liquid analysis (pneumatic nebulization, droplet generation) and elemental imaging by laser ablation ICP-MS (LA-ICP-MS) of single cells are highlighted. Because of the high complexity of biological systems and for a better understanding of processes and dynamics of biologically or medically relevant cells, the authors discuss the idea of “multimodal spectroscopies.”     

Boron Determination—A Review of Analytical Methods

This paper reviews published methods of sample preparation, determinand purification, and the determination of boron concentration and isotopic composition in a sample. The most common methods for the determination of B concentration are spectrophotometric and plasma-source spectrometric methods. Although most spectrophotometric methods are based on colorimetric reactions of B with azomethine-H, curcumin, or carmine, other colorimetric and fluorometric methods have also been used to some extent. These methods, in general, suffer from numerous interferences and have low sensitivity and precision. Application of nuclear reaction and atomic emission/absorption spectrometric (AES/AAS) methods has remained limited because these methods have poor sensitivity and suffer from serious memory effects and interferences. Among a large number of published nuclear reaction methods only prompt-γ spectrometry has been of practical use. The prompt-γ method can determine B concentration in intact samples, which makes this method especially useful for some medical applications, including boron neutron capture therapy. However, this is a time-consuming method and not suitable for detection of low levels of B. Inductively coupled plasma optical emission spectrometry (ICP-OES) created a new dimension in B determination because of its simplicity, sensitivity, and multielement capability. However, it suffers interferences and is not adequately sensitive for some nutritional and medical applications involving animal tissues that are naturally low in B. All methods involving the measurement of B isotopic composition require a mass spectrometer. Thermal ionization mass spectrometry (TIMS) and secondary ion mass spectrometry (SIMS) have been used to measure isotopic composition of B; however, these methods are time consuming and require extensive sample preparation and purification. Development of inductively coupled plasma mass spectrometry (ICP-MS) not only overcame most of the drawbacks of earlier methods, but also its capabiltiy of measuring B isotopes made possible (1) B concentration determination by isotope dilution, (2) verification of B concentration by isotope fingerprinting in routine analysis, and (3) determination of total B concentration and B isotope ratio for biological tracer studies in the same run. Therefore, plasma source MS appears to be the method of choice among present-day technologies.     

Mass spectrometry as test bench for medicinal chemistry studies

This review describes how mass spectrometry can be used as a powerful test bench to obtain information on the biological activity of target compounds. Considering that mass spectrometry is based on the chemical reactivity of the analytes, it is possible to investigate the stability of the active compounds, to predict their behaviour in the environment of interest, and to obtain structure–reactivity relationships for new molecules of pharmacological interest. Electron ionization and metastable ion studies give evidence of the correlation between the mutagenic properties of a series of aryl and heteroaryl triazenes and mass spectrometric data. A linear relationship between the energetics of C(O)–O bond cleavage of some carbamic acid O-aryl esters and their FAAH inhibition activity has been proved by electrospray-ionization ion-trap mass spectrometry. An inverse correlation between the stability and cytotoxic activity of some copper complexes has been clearly established by electrospray-ionization mass spectrometry. Moreover, because of the sensitivity and specificity of mass spectrometry, it has been possible to determine and characterize impurities that in some cases can be the real bioactive compound.     

Characterization of proteins by mass spectrometry. Invited lecture.

Plasma desorption and fast atom bombardment mass spectrometry have in the last decade demonstrated the potential of mass spectrometry for protein studies. The recently developed matrix-assisted laser desorption and electrospray mass spectrometry have expanded the analytical potential of mass spectrometry to cover nearly all proteins. The type of information obtained with the four methods is described and their performances are compared. The potential of combining mass spectrometric relative molecular mass information on proteins with the information contained in protein sequence databases is outlined and some typical fields of application of mass spectrometry in protein chemistry are described. The need for the full integration of mass spectrometry in the protein laboratory is discussed.     

Distribution profiles of nitroxide spin probes in human skin—a combined study using spatially resolved electron spin resonance spectroscopy and mass spectrometry

Electron spin resonance spectroscopy and mass spectrometry are two analytical methods that are very rarely used in combination. In this paper, we will show that the methods complement one another in the example of the distribution of stable nitroxide radicals in human skin, including the spatial resolution of these distribution processes. There are many ESR investigations dealing with this subject, but unfortunately, they are all limited to the detection of paramagnetic species. The combination with MS allows the successful examination of the distribution profile of the main biotransformation product of the nitroxide radicals, the respective “ESR-silent” hydroxylamines. In order to maintain the biological state of the sample material as far as possible, atmospheric pressure matrix-assisted laser desorption/ionization with ion trap detection has been used for the mass spectrometric investigations. The results validate the former findings of the strong reduction of stable free radicals by biological material; moreover, the diamagnetic species formed during these processes have been identified.     

Mass spectrometric analysis of ceramic components for solid oxide fuel cells

For the determination of trace impurities in ceramic components of solid oxide fuel cells (SOFCs), some mass spectrometric methods have been applied such as spark source mass spectrometry (SSMS), laser ionization mass spectrometry (LIMS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and inductively coupled plasma mass spectrometry (ICP-MS). Due to a lack of suitable standard reference materials for quantifying of analytical results on La _x Sr _y MnO₃ cathode material a matrix-matched synthetic standard-high purity initial compounds doped with trace elements-was prepared in order to determine the relative sensitivity coefficients in SSMS and LA-ICP-MS. Radiofrequency glow discharge mass spectrometry (rf-GDMS) was developed for trace analysis and depth profiling of thick non-conducting layers. Surface analytical techniques, such as secondary ion mass spectrometry (SIMS) and sputtered neutral mass spectrometry (SNMS), were used to determine the element distribution on surfaces (homogeneity) and the surface contaminants of SOFC ceramic layers.Dedicated to Professor Dr. rer. nat. Hubertus Nickel on the occasion of his 65th birthday     

Identification of components in waste streams by electrospray and tandem mass spectrometry

Highly polar, non-gas-chromatographable compounds have few unambiguous analysis protocols for environmental applications. A recent environmental investigation, concerning the identification of a non-gas-chromatographable yellow component in chemical waste water and in effluents from a biological wastewater treatment plant required the use of a number of analytical approaches. Electrospray mass spectrometry, tandem mass spectrometry, high-performance liquid chromatography, nuclear magnetic resonance, and molecular spectroscopy of commercial and synthesized chlorodinitrophenol isomers were required in order to identify the specific isomer causing the color. The present report summarizes the electrospray ionization and tandem mass spectrometric studies that were used. The mass spectrometric study shows that two different isomers of chlorodinitrophenol exhibit very different collision-induced dissociation (CID) spectra. Differences in the tandem mass spectra can be attributed to the different structures of the anions formed from these two different isomers. Instrumentation that uses electrospray ionization and produces CID mass spectra and optical absorption spectra in a single analysis may be required in order to produce highly specific information on non-gas-chromatographable compounds found in the environment.     

Determination of fission products in nuclear samples by capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS)

Determination of fission products and of their isotopic composition is of high importance for the characterisation and complete inventory of nuclear fuels. Radiometric and mass spectrometric methods, which are currently used, enable only an incomplete determination of the fission products. Radiometric methods cannot be applied to all fission products and mass spectrometric methods are hindered by the existence of isobaric interferences, therefore a previous chemical separation is required before mass spectrometric analysis. Capillary electrophoresis (CE) has been coupled with inductively coupled plasma mass spectrometry (both ICP-QMS and ICP-SFMS). Typical detection limits of 6 ng/mL and 4 pg/mL for caesium as well as 8 ng/mL and 7 pg/mL for lanthanides have been obtained by CE-ICP-QMS and CE-ICP-SFMS, respectively. In addition to these very low detection limits, the procedures present the advantages to be fast (6 min for caesium and 13 min for lanthanides, respectively), to require a low microliter range sample volume and a nanoliter range injection volume. The radiation dose for the personnel as well as the volume of nuclear liquid wastes generated during the measurements are consequently reduced.The procedures have been applied to nuclear samples from PUREX process and leachates from MOX fuels.     

Trace and surface analysis of ceramic layers of solid oxide fuel cells by mass spectrometry

For the trace analysis of impurities in thick ceramic layers of a solid oxide fuel cell (SOFC) sensitive solid-state mass spectrometric methods, such as laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and radiofrequency glow discharge mass spectrometry (rf-GDMS) have been developed and used. In order to quantify the analytical results of LA-ICP-MS, the relative sensitivity coefficients of elements in a La(0.6)Sr(0.35)MnO(3) matrix have been determined using synthetic standards. Secondary ion mass spectrometry (SIMS) - as a surface analytical method - has been used to characterize the element distribution and diffusion profiles of matrix elements on the interface of a perovskite/Y-stabilized ZrO(2) layer. The application of different mass spectrometric methods for process control in the preparation of ceramic layers for the SOFC is described.     

面向DNA甲基化修饰分析的质谱技术

DNA甲基化作为表观遗传修饰中一种重要的调控方式,通过调控基因的表达,从而影响机体内一系列的生物学过程。色谱-质谱法是研究DNA甲基化修饰的重要研究手段。随着对哺乳动物DNA甲基化的生物学功能的深入研究,应用于研究表观遗传修饰的手段与仪器设备越来越先进。为了对DNA修饰进行定性与定量的分析检测,除了高效液相色谱整合不同种类质量分析器的质谱联用（HPLC-MS）技术外,目前还开发应用了基质辅助激光解析质谱技术（MALDI-ToF-MS）和气相色谱-质谱联用技术（GC-MS）,从而极大拓展了DNA甲基化修饰研究的手段。本文对分析表观遗传DNA甲基化修饰的质谱技术发展进行综述,希望为DNA甲基化修饰分析提供有价值的研究策略。     

Characterization of copolymers of polycarbonate and polydimethylsiloxane by 2D chromatographic separation,MALDI-TOF mass spectrometry,and FTIR spectroscopy

Abstract

The structure and composition of polycarbonate polydimethylsiloxane copolymer (PC-co-PDMS) was investigated by applying various analytical approaches including chromatographic separation methods, spectrometric, and spectroscopic detection techniques. In particular, size exclusion chromatography (SEC) and liquid adsorption chromatography operating at different conditions (e.g. using gradient solvent systems) were used to achieve separations according to molar mass and functionality distribution. The coupling of both techniques resulted in fingerprint two-dimensional plots, which could be used to easily compare different copolymer batches. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry was applied for structural investigations. The different ionization behavior of both comonomers, however, strongly limited the applicability of this technique. In contrast to that, Fourier-transform Infrared (FTIR) spectroscopy could be used to quantify the amount of PDMS in the copolymer at different points in the chromatogram. The resulting methodology was capable of distinguishing PC-co-PDMS copolymer from PC homopolymer chains present in the material.     

Photo-ionisation mass spectrometry as detection method for gas chromatography. Optical selectivity and multidimensional comprehensive separations

Mass spectrometry (MS) with soft ionisation techniques (i.e. ionisation without fragmentation of the analyte molecules) for gaseous samples exhibits interesting analytical properties for direct analysis applications (i.e. direct inlet mass spectrometric on-line monitoring) as well as mass spectrometric detection method for gas chromatography (GC-MS). Commonly either chemical ionisation (CI) or field ionisation (FI) is applied as soft ionisation technology for GC-MS. An interesting alternative to the CI and FI technologies methods are photo-ionisation (PI) methods. PI overcomes some of the limitations of CI and FI and furthermore add some unique analytical properties. The resonance enhanced multi-photon ionisation (REMPI) method uses intense UV-laser pulses (wavelength range approximately 350-193 nm) for highly selective, sensitive and soft ionisation of predominately aromatic compounds. The single photon ionisation (SPI) method utilises VUV light (from lamps or laser sources, wavelengths range approximately 150-110 nm) can be used for a universal soft ionisation of organic molecules. In this article the historical development as well as the current status and concepts of gas chromatography hyphenated to photo-ionisation mass spectrometry are reviewed.     

Assessment of the pollutant elimination efficiency by gas chromatography/mass spectrometry, liquid chromatography-mass spectrometry and -tandem mass spectrometry. Comparison of conventional and membrane-assisted biological wastewater treatment processes

The elimination efficiency of advanced conventional biological wastewater treatment was compared to membrane-assisted biological wastewater treatment. The sum parameter analyses dissolved organic carbon (DOC) and chemical oxygen demand (COD) or substance-specific analyses such as gas chromatography combined with mass spectrometry, flow injection analysis (FIA-MS) and liquid chromatography (LC-MS) in combination with mass or tandem mass spectrometry (MS-MS) were applied to assess elimination of hardly eliminable compounds in both types of wastewater treatment plants (WWTP). Reduction of DOC and COD in wastewater treatment processes confirmed a favourable elimination efficiency. Substance-specific methods which were applied in addition permitted a qualitative and semi-quantitative assessment of elimination with a visual pattern recognition approach. In order to identify pollutants either the NIST library of electron impact mass spectra for unpolar compounds or the laboratory-made collision-induced dissociation spectra library for polar pollutants was used. To assess elimination efficiency FIA-MS in the selected ion monitoring mode (SIM) besides high selective substance-specific mass spectrometric techniques such as parent ion scans and neutral loss scans were used for quantification. Results proved that membrane-assisted treatment was more effective than advanced biological treatment. In both types of WWTPs predominantly unpolar pollutants were eliminated, while all effluents were dominated by polar compounds of anthropogenic and biogenic origin. These unpolar and polar compounds which had been identified as hardly eliminable are reported about. Quantitative results obtained by FIA-MS, LC-MS and MS-MS for the elimination of alkyl polyglycol ethers, nonylphenol ethoxylates and linear alkylbenzenesulfonic acids from wastewater are presented.     

Direct High-Performance Liquid Chromatographic Enantiomer Separation of Anti-Inflammatory Planar Chiral [2.2]Paracyclophane-4-acetic Acid

Fifty years after the discovery of natural corticosteroid hormones and their anti-inflammatory properties many synthetic derivatives of these molecules are now available. Most are widely used in human and veterinary medicine, legally but under regulated conditions. These compounds can also be used as growth promoters in animal breeding, although such use is illegal in Europe. Consequently, analytical methods have been developed to monitor use of corticosteroids in cattle. This paper, based on the authors experience and the main relevant literature, describes the different mass spectrometric approaches used for measurement of corticosteroid residues (parent drug, metabolites, and esters) in biological matrices (urine, meat, hair), including gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS²). The respective advantages of liquid chromatography and gas chromatography, in conjunction with different derivatisation reactions, are discussed. The behavior of corticosteroids with different ionization techniques is also discussed. Application to monitoring corticosteroid misuse and to investigation of pharmacokinetics and metabolism in bovine species is described and new data are presented relating to elimination and hair fixation kinetics for free and ester forms and the nature and proportions of corticosteroid phase I and phase II metabolites. Finally, this work reviews ten years experience of the use of a variety of mass spectrometric techniques for analysis of corticosteroids in animals produced as food.