Ionization techniques in capillary electrophoresis-mass spectrometry: principles, design, and application

A major step forward in the development and application of capillary electrophoresis (CE) was its coupling to ESI-MS, first reported in 1987. More than two decades later, ESI has remained the principal ionization technique in CE-MS, but a number of other ionization techniques have also been implemented. In this review the state-of-the-art in the employment of soft ionization techniques for CE-MS is presented. First the fundamentals and general challenges of hyphenating conventional CE and microchip electrophoresis with MS are outlined. After elaborating on the characteristics and role of ESI, emphasis is put on alternative ionization techniques including sonic spray ionization (SSI), thermospray ionization (TSI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), matrix-assisted laser desorption ionization (MALDI) and continuous-flow fast atom bombardment (CF-FAB). The principle of each ionization technique is outlined and the experimental set-ups of the CE-MS couplings are described. The strengths and limitations of each ionization technique with respect to CE-MS are discussed and the applicability of the various systems is illustrated by a number of typical examples.     

Atmospheric pressure photoionization mass spectrometry

Atmospheric pressure photoionization (APPI) is the last arrival in the family of atmospheric pressure ionization (API) methods to couple mass spectrometry (MS) to liquid-phase separation techniques. The basic idea was to further extend the fields of application of liquid chromatography (LC)-MS to those molecules that are not, or are poorly amenable, to electrospray (ESI) or APCI. The present review explores the literature. After a short introduction with an historical background and the premises for its development, we describe the technique, its physical principles, and the factors that affect its efficiency. The review also presents a survey of applications in different fields.     

采用多重离子化方式探讨氨基酸类化合物的离子化效率

Diverse amino acids produce different signal responses with disparate ionization. Accordingly, none of the currently available mass spectrometric techniques can analyze all amino acids simultaneously. Here, it was a better way that combination of electrospray ionization(ESI), atmospheric pressure chemical ionization(APCI) and atmospheric pressure photo ionization(APPI) enhanced ionization efficiency for amino acids. The results show that the application of multiple ionization mass spectrometry is expected to provide a powerful new tool for amino acids research.     

Characterization of harmonics and multi-charged peaks obtained by Fourier transform ion cyclotron resonance mass spectrometry

The differences between harmonics and multi-charged peaks in mass spectrometry are often not obvious. This work conducts experiments using both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to investigate the difference between harmonics and multi-charged peaks for the first time. In particular, organometallic compounds, which were characterized by the isotope distribution in ESI, were investigated in detail. A comparison of the peaks of the three charges with the three frequency doubling results at high-resolution mass spectra demonstrated that these peaks may be clearly differentiated from one another. Fullerene (C₆₀) was characterized using APCI in the negative mode by FTICR MS provided further evidence. In addition, harmonics are unavoidable, but can be relatively weakened. These results are helpful to obtain accurate and comprehensive spectral information from FTICR MS.     

大气压电离液质联机的应用

本文以我们几年来的实际工作为素材，对电喷雾（ESI）和大气压化学电离（APCI）这一近年发展起来的软电离质谱接口技术做了应用性介绍。文中列举了该技术用于药物代谢、蛋白质构象以及热不稳定化合物分析的几个实例，对一些典型化合物在离子源内经碰撞诱导解离（CID）形成的质谱开裂产物进行了解析，对流量匹配、脱溶剂的技术特点、离子在溶液中的预形成、溶剂的选择、仪器的噪声等实践中遇到的问题做了较为详细的讨论。     

Mass spectrometry imaging under ambient conditions

Mass spectrometry imaging (MSI) has emerged as an important tool in the last decade and it is beginning to show potential to provide new information in many fields owing to its unique ability to acquire molecularly specific images and to provide multiplexed information, without the need for labeling or staining. In MSI, the chemical identity of molecules present on a surface is investigated as a function of spatial distribution. In addition to now standard methods involving MSI in vacuum, recently developed ambient ionization techniques allow MSI to be performed under atmospheric pressure on untreated samples outside the mass spectrometer. Here we review recent developments and applications of MSI emphasizing the ambient ionization techniques of desorption electrospray ionization (DESI), laser ablation electrospray ionization (LAESI), probe electrospray ionization (PESI), desorption atmospheric pressure photoionization (DAPPI), femtosecond laser desorption ionization (fs‐LDI), laser electrospray mass spectrometry (LEMS), infrared laser ablation metastable‐induced chemical ionization (IR‐LAMICI), liquid microjunction surface sampling probe mass spectrometry (LMJ‐SSP MS), nanospray desorption electrospray ionization (nano‐DESI), and plasma sources such as the low temperature plasma (LTP) probe and laser ablation coupled to flowing atmospheric‐pressure afterglow (LA‐FAPA). Included are discussions of some of the features of ambient MSI for example the ability to implement chemical reactions with the goal of providing high abundance ions characteristic of specific compounds of interest and the use of tandem mass spectrometry to either map the distribution of targeted molecules with high specificity or to provide additional MS information on the structural identification of compounds. We also describe the role of bioinformatics in acquiring and interpreting the chemical and spatial information obtained through MSI, especially in biological applications for tissue diagnostic purposes. Finally, we discuss the challenges in ambient MSI and include perspectives on the future of the field. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:218–243, 2013     

Recent advances in mycotoxin determination in food and feed by hyphenated chromatographic techniques/mass spectrometry

Mycotoxins are fungal toxins produced by molds, which occur universally in food and feed derivatives, and are produced under certain environmental conditions in the field before harvest, post-harvest, during storage, processing, and feeding. Mycotoxin contamination is one of the most relevant and worrisome problem concerning food and feed safety because it can cause a variety of toxic acute and chronic effects in human and animals. In this review we report the use of mass spectrometry in connection with chromatographic techniques for mycotoxin determination by considering separately the most diffuse class of mycotoxins: patulin, aflatoxins, ochratoxin A, zearalenone, trichothecenes, and fumonisins. Although the selectivity of mass spectrometry is unchallenged if compared to common GC and LC detection methods, accuracy, precision, and sensitivity may be extremely variable concerning the different mycotoxins, matrices, and instruments. The sensitivity issue may be a real problem in the case of LC/MS, where the response can be very different for the different ionization techniques (ESI, APCI, APPI). Therefore, when other detection methods (such as fluorescence or UV absorbance) can be used for the quantitative determination, LC/MS appears to be only an outstanding confirmatory technique. In contrast, when the toxins are not volatile and do not bear suitable chromophores or fluorophores, LC/MS appears to be the unique method to perform quantitative and qualitative analyses without requiring any derivatization procedure. The problem of exact quantitative determination in GC/MS and LC/MS methods is particularly important for mycotoxin determination in food, given the high variability of the matrices, and can be solved only by the use of isotopically labeled internal standards or by the use of ionization interfaces able to lower matrix effects and ion suppressions. When the problems linked to inconstant ionization and matrix effects will be solved, only MS detectors will allow to simplify more and more the sample preparation procedures and to avoid clean-up procedures, making feasible low-cost, high-throughput determination of mycotoxins in many different food matrices.     

Mass spectrometric methods for monitoring redox processes in electrochemical cells

Electrochemistry (EC) is a mature scientific discipline aimed to study the movement of electrons in an oxidation–reduction reaction. EC covers techniques that use a measurement of potential, charge, or current to determine the concentration or the chemical reactivity of analytes. The electrical signal is directly converted into chemical information. For in‐depth characterization of complex electrochemical reactions involving the formation of diverse intermediates, products and byproducts, EC is usually combined with other analytical techniques, and particularly the hyphenation of EC with mass spectrometry (MS) has found broad applicability. The analysis of gases and volatile intermediates and products formed at electrode surfaces is enabled by differential electrochemical mass spectrometry (DEMS). In DEMS an electrochemical cell is sampled with a membrane interface for electron ionization (EI)‐MS. The chemical space amenable to EC/MS (i.e., bioorganic molecules including proteins, peptides, nucleic acids, and drugs) was significantly increased by employing electrospray ionization (ESI)‐MS. In the simplest setup, the EC of the ESI process is used to analytical advantage. A limitation of this approach is, however, its inability to precisely control the electrochemical potential at the emitter electrode. Thus, particularly for studying mechanistic aspects of electrochemical processes, the hyphenation of discrete electrochemical cells with ESI‐MS was found to be more appropriate. The analytical power of EC/ESI‐MS can further be increased by integrating liquid chromatography (LC) as an additional dimension of separation. Chromatographic separation was found to be particularly useful to reduce the complexity of the sample submitted either to the EC cell or to ESI‐MS. Thus, both EC/LC/ESI‐MS and LC/EC/ESI‐MS are common. © 2013 The Authors. Mass Spectrometry Reviews published by Wiley Periodicals, Inc. Mass Spec Rev 34:64–92, 2015     

Characterization of proteins by ambient mass spectrometry

Proteins play important roles in living systems and are topics of many fundamental and applied research projects. With the introduction of electrospray ionization and matrix‐assisted laser desorption/ionization for analysis of biomacromolecules in the late 1980s, mass spectrometry has become an important tool for characterization of proteins. Characterization of proteins in raw samples by these mass spectrometric techniques, however, usually requires extensive sample pretreatment. Ambient ionization techniques are new mass spectrometric techniques that allow direct analysis of samples with no or little sample preparation. Can these techniques facilitate or even eliminate sample preparation for mass spectrometric analysis of proteins? Apart from sample preparation, do these techniques offer any new features for characterization of proteins as compared with conventional ESI or MALDI? Recent advances in characterization of proteins by ambient mass spectrometry are summarized and commented in this article. © 2011 Wiley Periodicals, Inc. Mass Spec Rev 31:437–447, 2012     

Phospholipids as cancer biomarkers: Mass spectrometry‐based analysis

Lipids, particularly phospholipids (PLs), are key components of cellular membrane. PLs play important and diverse roles in cells such as chemical‐energy storage, cellular signaling, cell membranes, and cell–cell interactions in tissues. All these cellular processes are pertinent to cells that undergo transformation, cancer progression, and metastasis. Thus, there is a strong possibility that some classes of PLs are expected to present in cancer cells and tissues in cellular physiology. The mass spectrometric soft‐ionization techniques, electrospray ionization (ESI), and matrix‐assisted laser desorption/ionization (MALDI) are well‐established in the proteomics field, have been used for lipidomic analysis in cancer research. This review focused on the applications of mass spectrometry (MS) mainly on ESI‐MS and MALDI‐MS in the structural characterization, molecular composition and key roles of various PLs present in cancer cells, tissues, blood, and urine, and on their importance for cancer‐related problems as well as challenges for development of novel PL‐based biomarkers. The profiling of PLs helps to rationalize their functions in biological systems, and will also provide diagnostic information to elucidate mechanisms behind the control of cancer, diabetes, and neurodegenerative diseases. The investigation of cellular PLs with MS methods suggests new insights on various cancer diseases and clinical applications in the drug discovery and development of biomarkers for various PL‐related different cancer diseases. PL profiling in tissues, cells and body fluids also reflect the general condition of the whole organism and can indicate the existence of cancer and other diseases. PL profiling with MS opens new prospects to assess alterations of PLs in cancer, screening specific biomarkers and provide a basis for the development of novel therapeutic strategies. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:107‐138, 2018     

Matrix-free methods for laser desorption/ionization mass spectrometry

Matrix assisted laser desorption/ionization (MALDI) is a soft ionization mass spectrometric method that has become a preeminent technique in the analysis of a wide variety of compounds including polymers and proteins. The main drawback of MALDI is that it is difficult to analyze low molecular weight compounds (<1,000 m/z) because the matrix that allows MALDI to work interferes in this mass range. In recent years there has been considerable interest in developing laser desorption/ionization (LDI) techniques for the analysis of small molecules. This review examines the approaches to matrix-free LDI mass spectrometry including desorption/ionization on silicon (DIOS), sol-gels, and carbon-based microstructures. For the purposes of this review matrix-free methods are defined as those that do not require matrix to be mixed with the analyte and therefore does not require co-crystallization. The review will also examine mechanisms of ionization and applications of matrix-free LDI-MS.     

Microchip technology in mass spectrometry

Microfabrication of analytical devices is currently of growing interest and many microfabricated instruments have also entered the field of mass spectrometry (MS). Various (atmospheric pressure) ion sources as well as mass analyzers have been developed exploiting microfabrication techniques. The most common approach thus far has been the miniaturization of the electrospray ion source and its integration with various separation and sampling units. Other ionization techniques, mainly atmospheric pressure chemical ionization and photoionization, have also been subject to miniaturization, though they have not attracted as much attention. Likewise, all common types of mass analyzers have been realized by microfabrication and, in most cases, successfully applied to MS analysis in conjunction with on‐chip ionization. This review summarizes the latest achievements in the field of microfabricated ion sources and mass analyzers. Representative applications are reviewed focusing on the development of fully microfabricated systems where ion sources or analyzers are integrated with microfluidic separation devices or microfabricated pums and detectors, respectively. Also the main microfabrication methods, with their possibilities and constraints, are briefly discussed together with the most commonly used materials. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:351‐391, 2010     

Mass spectrometric characterization of high-valent metal-oxo, -peroxo and -peroxy intermediates of relevance in oxidation processes

The coupling of mass spectrometry with ionization techniques like electrospray ionization (ESI) or matrix-assisted matrix-assisted laser desorption-ionization (MALDI) offers many advantages over other well-established spectroscopic techniques employed for the investigation of intermediates or short-lived species in condensed-phase. In this review we describe some of the applications of mass spectrometry, in particular of ESI-MS to the detection and characterization of high-valent metal-oxo, -peroxo and -peroxy derivatives, crucial intermediates in the oxyfunctionalization of organic substrates. In addition, by utilizing gas-phase ion-molecule reactions and MS/MS experiments, information on the intrinsic reactivity of the short-lived intermediates may be obtained. The combined use of ESI-MS in association with other spectroscopic techniques and theoretical calculations is discussed as well.     

Mass spectrometry in the biosynthetic and structural investigation of lignins

Lignin, a resistant cell-wall constituent of all vascular plants that consists of ether and carbon-linked methoxyphenols, is still far from being structurally described in detail. The main problem in its structural elucidation is the difficulty of isolating lignin from other wood components without damaging lignin itself. Furthermore, the high number and variegated forms of linkages that occur between the monomeric units and the chemical resistance of certain ether bonds limit the extent to which analytical and degradation procedures can be used to elucidate the lignin structure. Most of our present knowledge about the molecular structure of lignin is based on the analysis of monomers, dimers or, at the most, tetramers of degraded isolated lignins. Mass spectrometry (MS), which offers advantages in terms of speed, specificity, and sensitivity, has revealed to be a very powerful technique in the structural elucidation of lignins, in combination with the great number of chemical and thermal degradation methods available in the study of lignin. Moreover, the recent development of new ionization techniques in MS-electrospray ionization (ESI)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS-has provided new possibilities to also analyze the undegraded lignin macromolecule.     

多氯联苯的质谱裂解规律探析

分析比较了6种多氯联苯（PCBs）化合物在电子轰击离子源（EI）和大气压化学离子源（APCI）条件下的质谱裂解规律。在APCI离子源中,无（准）分子离子峰,但存在质量比化合物分子质量少19 u的加合离子峰,此加合离子同位素峰的强度变化规律与EI质谱中分子离子同位素峰的强度变化规律一致;结合不同流动相辅助探讨此类物质的裂解途径,推断该加合离子是多氯联苯解离一个氯原子后从流动相的水分子中捕获一个羟基,随后又失去一个质子形成的。该质谱裂解规律有助于为此类化合物的快速鉴定提供依据。     

A tutorial in small molecule identification via electrospray ionization‐mass spectrometry: The practical art of structural elucidation

The identification of unknown molecules has been one of the cornerstone applications of mass spectrometry for decades. This tutorial reviews the basics of the interpretation of electrospray ionization‐based MS and MS/MS spectra in order to identify small‐molecule analytes (typically below 2000 Da). Most of what is discussed in this tutorial also applies to other atmospheric pressure ionization methods like atmospheric pressure chemical/photoionization. We focus primarily on the fundamental steps of MS‐based structural elucidation of individual unknown compounds, rather than describing strategies for large‐scale identification in complex samples. We critically discuss topics like the detection of protonated and deprotonated ions ([M + H]⁺ and [M ? H]^?) as well as other adduct ions, the determination of the molecular formula, and provide some basic rules on the interpretation of product ion spectra. Our tutorial focuses primarily on the fundamental steps of MS‐based structural elucidation of individual unknown compounds (eg, contaminants in chemical production, pharmacological alteration of drugs), rather than describing strategies for large‐scale identification in complex samples. This tutorial also discusses strategies to obtain useful orthogonal information (UV/Vis, H/D exchange, chemical derivatization, etc) and offers an overview of the different informatics tools and approaches that can be used for structural elucidation of small molecules. It is primarily intended for beginning mass spectrometrists and researchers from other mass spectrometry sub‐disciplines that want to get acquainted with structural elucidation are interested in some practical tips and tricks.

     

Discovering new invertebrate neuropeptides using mass spectrometry

Neuropeptides are a complex set of messenger molecules controlling a wide array of regulatory functions and behaviors within an organism. These neuromodulators are cleaved from longer protein molecules and often experience numerous post-translational modifications to achieve their bioactive form. As a result of this complexity, sensitive and versatile analysis schemes are needed to characterize neuropeptides. Mass spectrometry (MS) through a variety of approaches has fueled the discovery of hundreds of neuropeptides in invertebrate species in the last decade. Particularly successful are direct tissue and single neuron analyses by matrix-assisted laser desorption/ionization (MALDI) MS, which has been used to elucidate approximately 440 neuropeptides, and examination of neuronal homogenates by electrospray ionization techniques (ESI), also leading to the characterization of over 450 peptides. Additional MS methods with great promise for the discovery of neuropeptides are MS imaging and large-scale peptidomics studies in combination with a sequenced genome.     

基质辅助激光解吸电离-飞行时间质谱在糖类化合物研究中的应用

综述了基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF-MS)的发展、在糖类化合物结构研究时常选用的基质,以及在不同类型糖化合物分析中的应用。MALDI-TOF-MS在糖类分析中通常采用的是N2激光源,基质多为有机小分子如2,5-二羟基苯甲酸、2,4,5-三羟基苯乙酮、1-羟基异喹啉或2-羟基-5-甲氧基苯甲酸、α-氰基-4-羟基-苯丙烯酸等,基质类型的选择则要取决于糖类的存在形式。糖类化合物如中性糖、酸性糖、硫酸化糖、糖蛋白、蛋白聚糖及糖脂等均可利用适合的基质而进行MALDI-TOF-MS分析。     

板蓝根化学成分及抗氧化活性的研究

采用高效液相色谱-串联质谱法（HPLC-MS/MS）和直接进样的电喷雾串联质谱法（ESI-MS/MS）分析板蓝根乙酸乙酯提取物、正丁醇提取物和95%乙醇提取物的化学成分。同时,采用铁离子还原能力法（FRAP）评价板蓝根的抗氧化活性,并通过自由基清除能力法（DPPH）对FRAP法的测试结果进行验证。结果表明：在板蓝根乙酸乙酯提取物、正丁醇提取物和95%乙醇提取物中共鉴别出吲哚类、喹唑酮类、有机酸类、核苷类、嘌呤类、氨基酸类、黄酮类以及糖类等28个化合物。研究发现,板蓝根抗氧化活性部位主要集中在极性较低的化学组分中,吲哚类、喹唑酮类和有机酸类化合物是其主要的有效成分。该方法简便、快捷、灵敏,可为中药化学成分分析和抗氧化活性评价提供方法参考。     

大青叶化学成分及抗氧化活性研究

采用高效液相色谱-大气压化学电离质谱法（HPLC-APCI/MS）和直接进样的电喷雾串联质谱法（ESI-MS²）分析大青叶乙酸乙酯、正丁醇和95%乙醇提取物的化学成分。采用Kromasil C18分析柱（4.6 mm×250 mm×5 μm），以0.15%乙酸/水-甲醇为流动相进行线性梯度洗脱，流速0.8 mL/min，柱温25 ℃；采用全波长扫描紫外检测器,检测波长分别为254 nm和265 nm；采用大气压化学电离源和电喷雾电离源，在正、负两种离子模式下进行检测。结果表明，在大青叶乙酸乙酯、正丁醇和95%乙醇提取物中共鉴别出39种化合物，包括生物碱类、有机酸类、核苷嘌呤类、氨基酸类、黄酮类、糖类、卟啉类以及含硫类成分化合物。采用铁离子还原能力法（FRAP）对大青叶的抗氧化活性进行评价，并用自由基清除能力法验证FRAP法的测试结果。抗氧化活性评价结果表明，大青叶抗氧化活性部位主要集中在极性较低的化学组分中，吲哚类、奎唑酮类和有机酸类化合物是其主要有效成分。该方法快捷、灵敏，适用于中药化学成分分析和抗氧化活性评价。