Overview Of Reaction Monitoring By Mass Spectrometry Biology Essay

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The elucidation of reaction mechanisms in the field of inorganic and organic chemistry, and more recently in the field of supramolecular chemistry, has attracted researchers' intensive interests. A better understanding of these reacting/assembling mechanisms will enable people to work more wisely and innovatively in designing new reactions and synthesizing novel chemicals and materials. Because of its high sensitivity, specificity and scanning speed, mass spectrometers (MS) are widely used in this area, which can directly and dynamically give the molecular mass information of reactants, reaction products and reaction intermediates, providing useful information for the study of reaction kinetics and mechanism. In this review, we categorize MS reaction monitoring techniques based on the ion source used. Especially noteworthy is the rapid development of ambient ion sources in the last decade which greatly facilitate the direct and rapid analysis of samples with no/little preparation. These ion sources, including desorption electrospray ionization (DESI), extractive electrospray ionization (EESI), low temperature plasma (LTP) probe, electrospray-assisted laser desorption/ionization (ELDI), and desorption/ionization on porous silicon (DIOS) among many others, offer varied reaction times intrinsically controlled by the corresponding experimental parameters. In this review, examples of different ion sources coupled with MS for reaction monitoring are discussed and the unique applications of specific ion sources are highlighted. It is believed that with this abundant toolbox of ion sources at hand one can be more confident in choosing the suitable method to effectively address the problem of interests.

Keywords: reaction monitoring, mass spectrometry, online and real-time, ion source

1. Introduction

Most chemical reactions proceed via reactive intermediates through a complicated sequence of reaction steps. A better understanding of the details of these steps will enable chemists to more efficiently control chemical reactions and to find the optimum reaction conditions. The invention of electrospray ionization (ESI), among a number of recently emerged ion sources especially ambient ion sources, has greatly expanded the application scale of mass spectrometry (MS) in the fields of inorganic and organometallic chemistry. The mass-to-charge ratios (m/z) of ions and their corresponding isotopic patterns acquired by ESI-MS in the monitoring process are valuable clues for intermediate structure analysis and mechanism study, which creates a new pathway for the investigation of organic reaction mechanisms. Through study of the evolution of critical reactive intermediate ions in the reacting process, important information related to reaction details can be acquired, which is otherwise unavailable by other research tools.

Up to now, a number of chemical and physical techniques have been employed to study reactive intermediates in organic chemical reactions. ESR spectroscopy, UV spectroscopy and in some appropriate cases chemically induced dynamic nuclear polarization (CIDNP) have been used for the direct detection of radical intermediates. Transient carbocations that contain a suitable chromosphore were also detected by UV spectroscopy. However, in general these reaction monitoring methods do not offer the capability for the direct and simultaneous detection of substrates, intermediates, and reaction products from reaction solutions. By contrast, ESI-MS is more universal in that most polar compounds can be ionized in a "soft" way inducing almost no fragmentation. Combined with the high scanning and analysis speed of a modern mass spectrometer, one can clearly find out that ESI-MS is a very simple method for the direct and simultaneous monitoring of substrates and reaction products formed, especially reactive intermediates.

Traditionally, reaction monitoring by ESI-MS is done by the so-called off-line approach: an aliquot of reaction solution is taken from an ongoing reaction system, diluted to stop the reaction and make the final solution suitable for MS analysis, and then introduced into MS. The reason for the dilution of the reaction solution rests with the fact that MS is a highly sensitive instrument and only a small amount of analyte is needed in order to be analyzed. Such a feature makes MS very suitable to be coupled with microfluidic devices to realize high-throughput screening of chemical reactions that proceed in very small volumes, in a more efficient, green, and environmentally friendly approach. Offline monitoring by ESI-MS is in many cases efficient and serves the purpose of reaction monitoring. However, in such cases as very fast reactions or reactions involving very short-lived reactive intermediates, offline ESI-MS monitoring (time resolution: ~ 0.2~1 min) is proved to be ineffective and monitoring methods with a higher time resolution are required. The booming of ambient ion sources following the invention of DESI in 2002, among many others including desorption/ionization on porous silicon (DIOS), electrospray-assisted laser desorption ionization (ELDI), extractive electrospray ionization (EESI), and low temperature plasma (LTP) probe, offers one many more choices in this aspect. These novel ion sources facilitate direct ambient sample analysis through elimination of sample preparation to make it possible to perform analyses under ambient conditions. Meanwhile, the field of reaction monitoring also benefits from such a rapid development in ion sources to address more challenging problems.

Reaction monitoring also has important applications in supramolecular chemistry in that it enables chemists working in this field to gain insights into the dynamics of self-assembling processes, which is very rarely known. In this review, we overview the currently available reaction monitoring methods based on MS. In particular examples of ion sources coupled with MS for reaction monitoring are discussed and the unique applications of some ambient ion sources are highlighted.

Figure 1. The number of publications related to reaction monitoring by mass spectrometry from 2000 to 2010. The search string used in the "topic" field was "(reaction monitor* OR reaction intermediate* OR reaction mechanism OR real-time monitor* OR online monitor) AND mass spec*". Data was retrieved from ISI web of science.

Table 1. MS techniques for reaction monitoring listed in order of publication



Year reported

Scope of applicability

Electron impact


Chemical ionization


Electrospray ionization


Direct analysis in real time


Matrix assisted laser desorption ionization


Membrane introduction mass spectrometry


Electrospray-assisted laser desorption/ionization


Extracted electrospray ionization


Desorption/ionization on silicon


Dielectric barrier discharge ionization


2. Reaction monitoring by ESI

Figure 2. Schematic diagram of the operation of an ESI needle in the positive ion mode.

The schematic diagram of an ESI needle which is operated in the positive ion mode was illustrated in Figure 2. As a versatile soft ionization technique, ESI has found wide applications in both offline and online reaction monitoring. Those applications that are performed in an offline approach require extraction of a small amount of mixture from the system of an ongoing reaction at a series of time points. Because ESI is especially suitable for the detection of small molecules, high-molecular-weight biomolecules such as biological polymers, proteins and peptides, and even relatively unstable noncovalent complexes, it is very useful in the studies of interactions between proteins and drugs , proteins and aptamers , and antigen-antibody combination . To use ESI for online and real-time reaction monitoring, Metzger and coworkers devised an online microreactor to have two reagents pumped through two separate tubings mixed, as shown in Figure 3. Capillaries and HPLC fittings are used to assemble such a mixer which offers a reaction time as short as 0.7 s. After mixing, the reaction can be monitored from the ESI spray capillary and under continuous flow conditions the mass spectra are acquired. To make the reaction time longer, one only need to use a longer ESI spray capillary. Using this device, they detected transient radical cations in electron-transfer-initiated Diels-Alder reactions , the catalytically active 14-electron ruthenium intermediate directly from solution , as well as homogeneously catalyzed Ziegler-Natta polymerization of ethene , among many other important organic reactions.

Figure 3. The on-line microreactor used for reagent mixing and subsequent reaction monitoring.

A prominent advantage of online ESI-MS analysis is that the delay between sampling and mass spectra display on the MS is eliminated. This allows the user to acquire an immediate profile of the ongoing reaction system, which is especially helpful for the monitoring of short-lived species, and useful in the elucidation of reaction mechanism. This aim is commonly fulfilled by stopped-flow ESI-MS. Shown in Figure 4 is the experimental setup of stopped-flow coupled with ESI-MS reported by Konermann and coworkers . Briefly, the plungers S1 and S2 are operated by computer-controlled drivers and are used to deliver the two reagents to the mixer M. When S1 and S2 are in operation, hard stoppers HS1 and HS2 are opened to allow flow of the fresh reagent mixture. The injection time of the mixture is 100 ms, for example. Afterwards, HS1 and HS2 are closed and plunger S3 is opened to deliver a 100 uL /min water flow to carry the "aliquot" of reagent mixture to the ESI-MS. The time resolution of this stopped-flow ESI-MS device is 2.5 s, in comparison with the millisecond time resolution of stopped-flow coupled with optical spectroscopy .

Figure 4. Experimental setup of stopped-flow ESI-MS . Notation: S1 and S2 syringes for pulsed sample injection; M mixer; S3 syringe. S3 delivers solvent that pushes reaction mixture through the fused silica capillary (C) to the ESI source after the reaction tube (R) has been filled with fresh reaction mixture; MS: quadrupole mass spectrometer; HS1, HS2: hard stoppers. Arrows indicate the directions of liquid flow.

In consideration of the particular importance to study the reaction kinetics of a wide range of chemical and biochemical systems, techniques which offer higher time resolution, i.e., seconds to milliseconds or even microseconds, are highly desirable. To reach the millisecond time resolution, Konermann and coworkers further proposed a capillary mixer with adjustable reaction chamber volume and achieved millisecond time resolution to study reaction dynamics in the "kinetic" mode. Figure 5A shows the schematic of this device . Figure 5B is a kinetic study of ubiquitin refolding using this reaction monitoring device by monitoring its molecular ions of four different charge states. Notice that the x scale (average reaction time) is within the range of 0~1.4 s and the ESI-MS intensity points plotted in the figure. It can be easily seen that the time resolution is high (millisecond scale).

Figure 5. (A) Schematic of a capillary mixer with adjustable reaction chamber volume. The high time resolution of this device is rationalized through adjustment of the reaction chamber between the capillary connected to syringe 1 and the ESI source. By gradually pulling back the capillary, chamber volume is increased from 0 mL and reaction time is also gradually increased from 0 s. (B) kinetic study of ubiquitin refolding by monitoring its molecular ions of four different charge states.

Following the above-mentioned online reaction monitoring techniques based on ESI, Plattner and coworkers explored the possibility of online ESI analysis of reactions under high pressures by making some improvements . Their experimental setup was shown in Figure 6.

Figure 6. Assembly of the online ESI analysis of reaction under high pressures. 1: high-pressure valve; 2: high-pressure split valve; 3: reactor assembly with fishing tube and sonotrode; 4: syringe for electrospray solvent; 5: ultrasonic piezoelectric transducer with horn; 6: mass spectrometer ion source; 7: fishing tube; 8, sample path and direction. (b) Reactor and MS assembly.

Briefly, CO2 was introduced into the reaction system via higher pressure valve 1 to maintain a high-pressure environment for the reaction to proceed. To record a mass spectrum, valve 2 was opened for 1 min to allow the reaction mixture to be driven out by the high pressure. The reaction mixture was then mixed with electrospray solvent delivered by syringe 4 to make the finally resulted solution suitable for ESI-MS analysis. Using this assembly, they monitored the dehydration of hydroxenin monoacetate and the hydrogenation of 5-norbornene-2-carbonitrile using Pd/CaCO3 as the catalyst. However, the time resolution of this work is not clearly described.

The above examples have already demonstrated that ESI-MS can be used to monitor a variety of chemical reactions. However, besides chemical reactions during which covalent bonds are formed, there are many other weak non-covalent interactions between molecules such as hydrogen bonding and π-π stacking. These weak forces are the driving force responsible for the association of molecules or assembly of them into regular structures. Researches have now realized the critical importance of understanding how these processes occur, as exemplified in very recent studies of real-time observation of the self-assembly of hybrid polyoxometalates and tracking of crown ether motion along a oligolysine chain , among many other early reports . However, complexes formed due to weak intermolecular interactions are very liable and some will even dissociate due to the relatively harsh conditions in conventional ESI-MS. In view of this, Yumaguchi and coworkers proposed the concept of cold spray ionization (CSI), a variant of ESI operated at ca. -80 ~10 C to allow facile characterization of liable organic species formed via non-covalent interactions. Shown in Figure 7 are the three typical CSI-MS setup. Up to now, CSI were used to study liable organometallic compounds including host-guest complexes, multiple-link interlocking complex, box-type complex, Grignard reagents and other organometallic compounds; as well as biomolecules such as proline aggregation, nucleosides, hyper-stranded DNAs. These progresses have been reviewed elsewhere and will not be detailed in this review.

Figure 7. CSI setup: (a), prototype; (b), axial type; (c), orthogonal type (for details, see reference ).

3. Reaction monitoring by DESI and SSI

The concept of DESI was proposed in the year of 2002 by Cooks and coworker aiming at providing the analytical community and beyond with an ambient MS analysis technique with little/no sample preparation . Since its invention, DESI-MS has found wide applications including the direct analysis of metabolites and drugs , explosives , as well as biological samples . This is to a large extent due to its simple assembly, fast sample introduction and overall robustness, in combination with the high sensitivity, selectivity and scanning speed of MS. An illustration of DESI is shown in Figure 8.

Figure 8. Illustration of DESI in the case high-throughput analysis of solid samples.

In DESI, applied the spray solvent is a DC electrical potential (methanol/water mixture is commonly used as the solvent). The solvent flows in the inner capillary tube and is nebulized by the nebulizing gas to generate small charged droplets. To assist solvent nebulization, a spray gas is flown through the space between the inner and outer capillaries which are concentric to each other. The linear velocity of the spray gas can reach as high as several hundred meters per second. The solid sample is then impacted by the charged droplets and the analytes contained within are picked up and ionized for MS analysis.

To get a better understanding of the mechanism of DESI, Cooks and coworkers used Computational fluid dynamics to model the atmospheric transport and droplet-thin film (surface) collisions in DESI and compared the experimental results with theoretical predictions, and they finally confirmed the "droplet-pickup" mechanism . A precondition of this work is that the sprayed droplets form a thin film on the solid surface which aids the transfer of the analytes from the solid phase into the gas phase. In view of this, Zhang's group envisaged that DESI may also be used to directly analyze liquid samples and developed a high-throughput DESI-MS analysis system . They demonstrated that the nebulizing gas used in DESI can also play the role of drawing liquid samples out of microfluidic channels, famously known as the Venturi effect, to form a thin layer, with the remaining processes similar to those in conventional DESI experiments in analyzing solid samples. Chen and coworkers went a further step: they coupled an electrochemical reaction cell with liquid-sampling DESI in an online approach . These and other reports consistently demonstrated that DESI is a reliable method in the analysis of liquid samples as well as the monitoring of ongoing solution reaction systems.

Very recently, Zare group reported an elegant work in which they succeeded in the detection of reaction intermediates on the millisecond time scale using DESI . One reagent (amino acid ligands) was added in the spray solution and the other ([{RuCl2(p-cymene)}2]) was deposited on surfaces. The very innovative aspect of this work is that the reaction time between these two reagents is intrinsically controlled by DESI to a few milliseconds in the secondary droplets. This argument can be rationalized by taking into consideration of the droplet velocity (~4 m/s) and the distance (0.5 cm) between the sample spot and the inlet of the MS. Under these experimental conditions, several reaction intermediates were detected and confirmed. ESI-MS can also reveal some reaction intermediates detected by DESI-MS, but there are also several other intermediates which are completely invisible in ESI mass spectra. The major parameter responsible for the contrast is the different reaction times in these two reaction monitoring methods: DESI on the time scale of only a few milliseconds while ESI on the time scale of 15 s. This example shows the very importance of reaction time control for reaction monitoring prior to MS analysis and strongly demonstrates that DESI is a very easy and suitable technique to detect short-lived intermediates of reactions that proceed or can be monitored in the ambient environment. Readers interested in this work are encouraged to read the original report .

4. Reaction monitoring by EESI

Figure 9. illustrates the schematic diagram of EESI . The name of the ionization technique depicts its specific features: by interaction of the ionizing spray (ESI spray) and the sample spray, analytes in the sample can be "extracted" into the ionization plume where charges are transferred to them to make them ionized. In comparison with ESI, EESI offers long-time signal stability and better tolerance towards complex samples without sample preparation. Due to its non-destructive and safe sampling feature and the above-mentioned advantages, EESI has been employed in the analyses of perfumes , fruits , toothpaste and water samples as well as in living objects analysis and in vivo fingerprinting of nonvolatile compounds involved in human metabolism . As will be demonstrated later, EESI is uniquely useful in the characterization of complex viscous liquids.

Figure 9. Schematic diagram of EESI-MS. The ionizing spray interacts with the sample spray in front of the MS inlet to transfer charges to the analytes contained in the latter to make them ionized and subsequently detected.

Reaction monitoring and mechanism elucidation using EESI-MS has also been reported. Zenobi's group explored this by direct coupling of a three-necked flask, with EESI . As shown in Figure 10, sampling of the reaction mixture was performed by passing nitrogen gas through the headspace of the flask. One should be aware that in this work it is the headspace gas (rather than the bulk solution) that was monitored. The utility of the technique has been demonstrated by monitoring a Michael addition reaction. However, a major problem was that it may not faithfully reflect the actual changes in the bulk solution over time, even the headspace gas is presumed to be in equilibrium with the bulk solution and is rapidly flushed out and updated. The time resolution of EESI-MS is reported to be no more than 0.2 s in the experimental setup shown in Figure 9.

Figure 10. Schematic diagram of the EESI reaction monitoring setup

Subsequently, the same group explored the possibility of using EESI to directly analyze complex liquid samples of high viscosity . The unique usefulness of EESI in the analysis of as well as in the monitoring of ongoing reactions in such kind of systems rests with the phenomenon called microjetting (Figure 11). Microjetting is a process during which the bubbles generated in the bulk liquid due to the introduction of nitrogen gas from EESI will burst at the liquid-air interface, resulting in the creation of microdroplets. Since EESI has been reported to analyze aerosols, it should be also be applicable in the analysis of these microdroplets, thereby providing rich chemical information of the complex viscous system. They successfully monitored the conversion of fructose to 5-hydroxymethylfurfural at 80 C in an ionic liquid, 1-ethyl-3-methylimidazolium chloride (EMIMCl), which is viscous, in an online and real-time approach. Liquids with a viscosity ranging from a few cP to 300, 000 can be rapidly characterized by this simple yet robust method with no sample preparation. The schematic illustration of the whole system is shown in Figure 10, which is similar to that shown in Figure 9.

Figure 11. Schematic illustration of the concept and setup of EESI-MS for the monitoring of ongoing chemical reactions in viscous liquid .

Besides, EESI can also provide new opportunities for the pathway elucidation of organic reactions which is difficult to achieve with other MS techniques. A mechanistic study shows that the ionization in EESI-MS occurs upon the intersection of the electrospray of a polar solvent and the neutral spray of the analyte through liquid-liquid extraction and charge transfer via microdroplets colliding, and this is also why this technique is termed as "extractive" ESI . Following this reasoning, it is reasonable to contemplate that reactions which proceed in the liquid phase can be repeated and monitored in a typical EESI-MS setup by adding reactants in the two sprays. Using this method, Metzger and coworkers monitored the electron-transfer-catalyzed (ETC) dimerization of trans-anethol to its dimerized product and confirmed a ring-closing process is involved in the reaction pathway, as illustrated in Figure 12.

Figure 12. ETC Dimerization of trans-anethol 2 to give 3 .

To study whether 3o•+ exists as an intermediate to transform into 3c•+ by undergoing a ring closing process, which is equivalent to determine whether 2 transforms into 3c•+ in a concerted approach, ESI-MS and EESI-MS analysis was carried out and the corresponding results were compared. Since 3c•+ and 3o•+ have the same mass to charge ratio (m/z 296), tandem MS experiments were performed to acquire the fragmentation patterns of these ions (m/z 296). Luckily, the fragmentation pattern of ions at m/z 296 by EESI-MS/MS was indeed different from that by ESI-MS/MS. To draw the conclusion, the EESI-MS/MS mass spectrum was compared with the CID (MS/MS) mass spectrum of authentic 3o, which was used as a standard compound. By this way, ions at m/z 296 in EESI-MS/MS mass spectrum were mainly attributed to 3o•+, and therefore the authors reached the conclusion that in the transformation of 2 to 3c•+, the two-step process rather than the one-step concerted process is supported by experimental results. The reason why 3o•+ could not be seen in ESI could be explained by the fast transformation of 2 to intermediate 3c•+ which can only be observed by EESI-MS that offers a time resolution of a few milliseconds.

5. Reaction monitoring by LTP probe

Sharing a same physics with dielectric barrier discharge ionization (DBDI), low temperature plasma (LTP) probe was developed in 2009 which is considered to be an improved version of DBDI in that it allows direct interaction of the plasma blown out of the discharge tube with the sample to be analyzed, facilitating convenient analysis of samples of any shape and size. Up to now, the LTP probe has been applied to a large variety of compounds including explosives , drug tablets and drugs of abuse , milk powders adulterated with melamine and non-destructive imaging of works of arts without any sample preparation. In consideration of its uniquely simple design and low temperature, Zhang's Group have tried to apply the LTP probe for direct monitoring of organic reactions in the ambient conditions and succeeded . Three classic organic reactions were selected as representative cases to illustrate the feasibility of the proposed method. The schematic diagram of the monitoring procedures is shown in Figure 13.

Figure 13. Schematic representation of the reaction monitoring procedures .

Reaction monitoring using a LTP probe may be more suitable for certain kinds of reactions than others, which depends on the method's sensitivity towards a specific category of compounds. Current studies show that the LTP probe in general offers a high sensitivity for compounds with a relatively high vapor pressure, such as esters and amines. But other experiments also revealed that explosives, the vapor pressures of which are very low, as well as active ingredients in tablets can also be easily detected. Therefore, the mechanism responsible for the desorption/ionization in the LTP probe is still vague and needs more supporting data to study it. We believe that with a better understanding of this mechanism, a whole category of chemical reactions can be effectively monitored by this simple yet convenient LTP probe in the ambient environment.

6. Reaction monitoring by ELDI, DIOS, and DART

Electrospray-assisted laser desorption/ionization (ELDI) combines the features of both ESI and laser desorption, which allows direct, sensitive, and rapid detection of both small organic molecules and large biological molecules . Basic ionization mechanism of ELDI includes release of neutral analyte molecules from the solid surfaces into the gaseous phase and subsequent ionization by charged species in the ESI plume. Unlike other ambient desorption/ionization techniques, desorption and ionization in ELDI are two separate processes. This feature offers independent control and optimization over the composition of the sample and the ESI plume. This ionization technique was proposed by Shiea's group and its application was subsequently demonstrated by the same group by coupling ELDI with thin layer chromatography (TLC) for continuous detection of separated chemical compounds without any sample preparation . In succession, it is observed that by adding carbon powders into the reaction system, ELDI can be utilized for direct analysis of liquid samples as well as for the direct and continuous monitoring of chemical reactions in the ambient conditions . The role of carbon powders is to transfer the energy of the laser beam to the solvent and analyte molecules, liberating them from the bulk system, and no viscous liquid medium as used in SALDI is needed. The schematic diagram of ELDI-MS for reaction monitoring is shown in Figure. 14. It is demonstrated that ELDI is suitable for continuous and online monitoring of chemical reactions including fast complexation reactions and relatively slow protein enzymolysis . However, though reaction monitoring was done in a continuously approach, the time resolution of this method was not reported.

Figure 14. Liquid ELDI system . (a) Schematic representation of the desorption and ionization of molecules dissolved in liquid during liquid ELDI. (b, c) Photos of the liquid ELDI system displaying (b) the electrospray plume and (c) mixing of analyte droplets with the electrospray plume.

Desorption/ionization on silicon (DIOS) is also a desorption MS technique. DIOS distinguishes itself from MALDI in that it employs no matrix to transfer the energy of the laser to the analytes . Instead of using small-molecule matrix, porous silicon is utilized to trap analytes, which also acts as the energy transfer medium to vaporize and ionize the trapped analytes upon laser irradiation. In comparison with MALDI, there is little interference in DIOS in the low molecular range arising from the matrix. Therefore, DIOS is also suitable for the analysis of small molecules. Siuzdak and coworkers, who invented DIOS, reviewed the development of this technique with respect to its application in small-molecule characterization, quantitative analysis, reaction monitoring, protein identification, and functional characterization of proteins .

As an example, the conversion of acetylcholine (ACh) to choline in the presence of acetylcholinesterase was monitored in a time scale of 250 min. Because DIOS chips were directly placed on a commercial MALDI plate, reaction monitoring is actually performed in an offline approach by shooting a laser beam to the sample at different time points. In this way, MS spectrum snapshots representative of the reaction progress were obtained. By extracting the intensities of reactants and/or reaction products from these MS spectra, the changes of their intensities over time, i.e. the conversion of ACh to choline, were reconstructed, as plotted in Figure 15d.

Figure 15. Configuration of the DIOS chip (a, b, c). a, On a MALDI plate four porous silicon plates are placed. Each of contains photopatterned spots/grids . b, Silicon-based laser desorption/ionization . c, Cross-section of the porous silicon as well as surface functionalities after hydrosilylation; R represents phenyl/alkyl chains . d. Plot of the conversion of ACh to choline at 25°C at an initial concentration of 200 mM substrate and a concentration of 40 pM enzyme .

7. Conclusions and perspectives

In this article, we have reviewed the applications of different reaction monitoring methods based on a variety of ion sources from the conventional ESI to the more recently emerged DESI, EESI, DIOS and LTP probe, etc. On the one hand, existing studies have demonstrated the great usefulness of conventional ion sources in the field of reaction monitoring, especially in reaction kinetics studies and interception of reaction intermediates for the elucidation of reaction mechanisms, and it is believed that ESI will continue to play a critical role in this fast-expanding field. On the other hand, reaction monitoring also benefits from the rapid development of ambient ion sources or novel sample introduction methods. For example, with the use of a simple DESI setup, interception of reaction intermediates on the milliseconds time scale is simple and straightforward since DESI intrinsically controls the reaction time to be a few milliseconds. Another example is the real-time monitoring of reactions by EESI without any sample pretreatment in a viscous liquid, which is impossible with any other existing MS method, owning to the interesting microjetting phenomenon. Therefore, the obstacles frequently encountered in ESI upon analysis of unusual systems can be overcome by using a novel ion source. The objective of this review is to give a brief introduction of the currently available methods and show especially their unique applications. As has been demonstrated, the large family of reaction monitoring methods based on MS offers one with much more flexibility and convenience in effectively addressing the problem of concern. It is believed that the rapid advancement of ion sources, combined with sample introduction techniques, will strongly drive the development of this very important field.