Mass SPectrometr Y catI ons F or A DvancIng MS AnaLYs Is ELectros PraY Mo DIFI DOI: 10.5702/massspectrometry.S0057

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Review

Electrospray Modifications for Advancing Mass Spectrometric Analysis Anil Kumar Meher and Yu-Chie Chen* Department of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan

Generation of analyte ions in gas phase is a primary requirement for mass spectrometric analysis. One of the ionization techniques that can be used to generate gas phase ions is electrospray ionization (ESI). ESI is a soft ionization method that can be used to analyze analytes ranging from small organics to large biomolecules. Numerous ionization techniques derived from ESI have been reported in the past two decades. These ion sources are aimed to achieve simplicity and ease of operation. Many of these ionization methods allow the flexibility for elimination or minimization of sample preparation steps prior to mass spectrometric analysis. Such ion sources have opened up new possibilities for taking scientific challenges, which might be limited by the conventional ESI technique. Thus, the number of ESI variants continues to increase. This review provides an overview of ionization techniques based on the use of electrospray reported in recent years. Also, a brief discussion on the instrumentation, underlying processes, and selected applications is also presented. Copyright © 2017 Anil Kumar Meher and Yu-Chie Chen. This is an open access article distributed under the terms of Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Please cite this article as: Mass Spectrom (Tokyo) 2017; 6(2): S0057 Keywords: electrospray, ionization, mass spectrometry, atmospheric pressure, ion source (Received September 23, 2016; Accepted December 30, 2016)

INTRODUCTION Since its development in 1980s, electrospray ionization mass spectrometry (ESI-MS) has been extensively used as a useful analytical tool in various research fields. The effect of electric field over water droplets has been known since the sixteenth century.1) In 1750, Jean-Antonie Nollet reported the earliest observation of electrospray phenomenon. However, it was more than two centuries before the term electrospray was used.2) Even before the success of coupling ESI with MS, the fundamental studies of electrospray process have been conducted. In 1917, Zeleny was the first one to observe the disintegration of liquid surface due to electric charges and reported the photographs of different spraying modes.3) Moreover, Dole et al.4) proposed the possibility to apply ESI coupled with a Faraday cup to determine the molecular weights of macromolecules through experimental observation and theoretical calculations. In 1984, Yamashita and Fenn proposed the first success result to couple ESI with MS.5,6) Later in 1988, Fenn and co-workers published two articles,7,8) which reported breakthrough in MS analysis results of macromolecules using ESI as the ionization method. In the first paper, the results showed the feasibility of using ESI-MS to analyze polymers wherein polyethylene glycol has an average molecular weight of 17500 Da.7) The second paper successfully demonstrated the suitability of using ESI-

MS for protein analysis.8) One crucial advantage of using electrospray to generate gas phase ions for macromolecules is that multiply-charged ions dominate the mass spectra. These ions are in a detectable m/z range in common mass analyzers. Thus, ESI-MS has been used as a useful analytical tool for characterization of small and large biomolecules9–11) since the middle of 1980s. In 2002, Fenn was awarded the Nobel Prize in Chemistry together with Tanaka and Kurt. As Fenn stated in his Nobel speech, “A few years ago, the idea of making proteins or polymers fly by ESI seemed as improbable as a flying elephant, but today it is a standard part of modern mass spectrometers.”12) This statement showed that the progress in the MS field is mainly resulting from those mass spectrometrists who can assert their beliefs and lead impossible to be possible. After 30 years since the first report of ESI-MS, this technique has also undergone various modifications. These modifications are mostly relied on the use of electrospray methods to explore new and simplified ionization techniques. Many open air ionization techniques based on the use of electrospray have been reported since the beginning of this century. Although commercial mass spectrometers are usually fitted with ESI or atmospheric pressure ionization source housed inside a chamber guarded with safety interlock, an ion source can also be directly operated in open air. The sprayer can be replaced with any design of ion source by incorporating an open interlock. Thus, all the commercial mass spectrometers

* Correspondence to: Yu-Chie Chen, Department of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan, e-mail: [email protected]

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have been exploited for coupling with almost all newly developed ionization methods. This flexibility has also allowed mass spectrometrists to discover unconventional ionization techniques.

ELECTROSPRAY-BASED IONIZATION TECHNIQUES In this review, we classify the ionization techniques based on modification of electrospray into two main categories: (1) modification of direct electrospray and (2) indirect application of electrospray for accomplishing ionization (Fig. 1). The first category is where the sample solution is mixed directly with electrospray solvent for ionization. Nevertheless, important parameters for generation of electrospray, including flow rate, pressure, sheath gas, and voltage, are varied for developing variants of electrospray-based ionization methods. In addition, some other techniques which use acoustics and specific substrates for generation of electrospray for MS analysis were also classified in this category. In the second category, ionization is not generated from the solution containing analytes and electrospray solvent. Alternatively, electrospray solvents and samples are separated and then merged through different means, such as fusion or collisions. That is, electrospray stream containing spray solvent only is used to interact with analytes prior to entering the inlet of a mass spectrometer.

Ionization techniques derived from modification of direct ESI source

Figure 1 shows the important parameters that can affect the generation of electrospray. On the basis of these parameters, several variants of electrospray-based ionization techniques have been explored. The details are discussed as follows.

Flow rate

The flow rate for directing sample solutions to the ESI emitter for ionization to occur has a tremendous effect on the ionization efficiency, which is increased with a decrease in sample flow rate. Ionization efficiency are reported to be improved at nanoliter flow rates.13,14) The improved ionization efficiency at such low flow rates is attributed to increased desolvation and increased charging efficiency occurring on per analyte molecule. This fact gave rise to the concept of nanoelectrospray for MS analyses. Formation of a stable nanoelectrospray is also dependent on many variables, including the fluid surface tension, solvent composition, conductivity of the fluid and the applied voltage and pressure.15) Wilm and Mann demonstrated that the flow

Fig. 1.

rate through an ESI capillary is highly correlated with the size of droplets formed from the capillary.13) On the basis of these findings, they developed a nanospray ionization technique, which can be used to generate electrospray using a very thin capillary (≤ 10 µm) with a very low flow rate of 20–40 nL/min.16) Nanoelectrospray ionization has been found to greatly reduce ion suppression and matrix effects, which typically occur in conventional ESI.17) These advantages have made nanoelectrospray ionization as a preferred choice for analysis of large biomolecules.18)

Pressure Subambient pressure ESI

Earlier, we discussed that ESI efficiency increases with the decrease of the flow rate of sample flow and can even attain 100% efficiency at the flow rate of nanoliter per minute when the analyte concentration is low. Nevertheless, this gain is limited due to ion losses during transmission from atmospheric pressure to the low pressure region of mass spectrometer.19,20) Analyte loss at the MS inlet is usually attributed to the dispersion of charged droplets to an area larger than the effective sampling area of inlet.20) Ion transmission between an ion source and the first vacuum stage is primarily dependent on the proximity and gas conductance at the interface.21) To improve the ion transmission, a patented design of ESI ion sources operating at low pressure was reported by Sheehan.22) In this setup, a liquid solution is introduced into a first vacuum chamber maintained at a reduced pressure of less than 0.1 Torr using a needle held at a high electric potential. The resultant liquid cone-jet is stabilized by an electrostatic lens which continues to propagate in axial direction towards the MS orifice. This setup allows almost all analyte in solution to be introduced through MS orifice.22) Smith et al. developed an optimized nanoESI method operated at subambient pressure, in which electrospray was generated inside a MS inlet with a chamber pressure of 30 Torr.20) Furthermore, they also modified the design to obtain a highest overall ionization efficiency of ∼50% at a lowest liquid flow rate of 50 nL/min.23) Nevertheless, evaporative cooling of the droplets disturbs the efficient gas phase ion formation via off-spring droplet fissions in vacuum ESI.

High pressure ESI

Pressurizing the ion source to a gas pressure greater than atmospheric pressure is a relatively new approach.24) A stable electrospray can be sustained for liquid with high surface tension under a super-atmospheric pressure environment.25) Super-atmospheric pressure ESI has been demonstrated with ESI26) as well as nanoESI.27,28) For such a high pressure ESI, the ion source consisting of an ESI emitter is placed in-

Classification of electrospray-based ionization methods. Page 2 of 11

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side a chamber filled with air or nitrogen gas. High pressure ESI is particularly useful when the spray solvent is completely aqueous and also when it is required to heat samples higher than 100°C.25)

Sheath gas

Ionization can also be achieved by employing gas flow with a sonic or supersonic speed without applying a voltage to generate spray. This ionization approach is called as sonic spray ionization (SSI) that was first reported by Hirabayashi et al.29,30) In conventional ESI, analytes acquire charges due to charge separation and subsequent disintegration of droplets induced by an electric field. In SSI, it is not required to generate spray for the formation of gas phase ions by directly applying an electric voltage under a high-speed gas flow of sonic spray processes. Hirabayashi and co-workers suggested two possible mechanisms: (1) microscopic fluctuations of ion concentrations in large droplets can induce an uneven distribution of positive and negative ions in the small droplets, which are formed by sudden disruptions of the large droplets. (2) The concentrations of negative and positive ions in the small droplets are different. When small droplets are generated from the surface of large droplets, the negative and positive ions are unevenly distributed.30) SSI has been successfully coupled with various other techniques such as capillary electrophoresis-MS31) and liquid chromatography (LC)-MS.32)

ESI Emitter Solid metal substrates

One of the earliest demonstrations for the application of solid substrate for electrospray generation was reported by Shiea and co-workers.33) They used a thin copper loop to load with sample solution thus electrospray was readily generated.33) The present study shed light on the possibility that ESI could be generated in a fast and simple way. Furthermore, the setup was modified by incorporating platinum instead of copper.34) The advantage of using platinum over copper is the chemical inertness of platinum which can prevent corrosion in regular ESI-MS analysis.34) That is, using inert platinum substrate can avoid oxidation because of the occurrence of electrochemical reactions. Shiea and co-workers also used a glass rod coated with a thin film of gold or Nafion for generation of electrospray.35) The coating rendered the surface with increased wettability for sufficient adherence of the sample on the substrate. Later, Hiraoka et al. reported a technique called probe electrospray ionization (PESI),36) which uses a stainless steel needle with micrometer-sized tip as the ESI emitter. PESI has been demonstrated to be useful in applications, such as clinical diagnosis,37) detection of illicit drugs in body fluids,38) reaction monitoring,39) and analysis of biological samples.40) Solvent droplets over a solid metal substrate with high voltage undergo a process similar to in traditional electrospray, in which a metal-made hollow capillary is used to direct sample solution and as the ESI emitter. However, in contrast to traditional electrospray, the metal capillary is not used and the metal surface is good enough for spray generation in PESI. One important highlight of PESI technique is that PESI is more tolerant to salts and detergents than capillarybased conventional ESI.41,42) In addition, Hu et al. demonstrated that commonly avail-

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able aluminium foils could also be used to assist the generation of electrospray.43) All the abovementioned techniques reiterate the fact that metal substrates are quite effective in assisting for generation of electrospray in an open air setup. Such approaches can be further extended to combine with other analytical techniques. For example, Pawliszyn and co-workers used a solid phase microextraction-like configuration for direct coupling of a metal substrate with MS. They used a stainless steel blade coated with a biocompatible polymer C18–polyacrylonitrile as the substrate.44) This technique, which is termed as coated blade spray, was demonstrated to have the capability to screen 21 compounds that are controlled by World Anti-Doping Agency and the United Nations Office on Drugs and Crime.44)

Porous materials

Most of the abovementioned methods used metallic probes for realization of electrospray because the probes have to be conductive in order to form electrospray. In contrast, common cellulose-based materials are not good electric-conductive substrates. However, their electrical conductivity could be significantly increased by adding a conducting medium, such as solvent.45) The concept of electrospray generation from porous material was first patented by Fenn in 1998.46) A wick element made of porous aggregates of fibers was used directly as the emitter. The sample liquid was supplied to the ESI ion source by a capillarity-induced flow through a wick element. This wick element is then comprised of aggregated fibers wetted by the sample liquid. Recently, various research groups have utilized substrates such as paper and wood as electrospray emitters.

Paper spray

Paper spray-MS was first described by Cooks and coworkers.47) Electrospray can be generated from the paper substrate placed in proximity to the orifice of a mass spectrometer (Fig. 2A) by loading a paper substrate with the mixture of analytes and spray solvent followed by applying with a high voltage. Paper spray has been applied in different fields such as bioanalysis,48) pharmacokinetics,49,50) forensics,51) microbiology,52,53) and food science.54,55) Recently, the technique has been commercialized by Prosolia under

Fig. 2. Schematic illustrations of (A) paper spray ionization and (B) contactless tissue paper assisted spray ionization. Page 3 of 11

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the trade name PaperSpray. Although paper spray offers several advantages, it has issues regarding ion suppression.56) Moreover, the paper strip is required to be directly connected to a high voltage. It is cumbersome to apply this method for high-throughput analysis. An alternative paper spray approach was reported recently to eliminate direct application of a high voltage over a paper substrate, in which electrospray was utilized by a high electric field provided by the mass spectrometer57) (Fig. 2B). Although the setup was simplified, this approach can be used for analysis of analytes through a wide mass range with a limit of detection as low as 10 −8 M.57) Additionally, there have been attempts to remove the necessity of a high voltage for generation of spray from paper. Pradeep et al. used a carbon nanotube impregnated filter paper for generating spray by application of voltage as low as 3 V.58) By using tellurium nanowire substrates, the voltage was further reduced to 1 V.59) This further led the work to explore ionization without any voltage at all. Wleklinski and co-workers demonstrated an approach of zero voltage paper spray by using chromatography paper.60) According to the suggested mechanism in zero voltage paper spray, the droplet experiences aerodynamic forces as it is pulled into the mass spectrometer by virtue of the suction pressure exerted by the vacuum system, leading to breakage of droplets into smaller droplets. During their journey through mass spectrometer inlet, the fine droplets are assumed to undergo multiple rounds of evaporation and Coulombic fission as occurring in traditional ESI.60) A similar work was also reported by Motoyama et al. at a similar time.61) Realizing the importance of such zero volt techniques, Cooks and co-workers recently patented the technique.62) Wide range of samples could not be analyzed with such zero volt paper spray approaches. However, these techniques bring us one step further for realizing the concept of portable mass spectrometers.

Wooden substrates

Using a wooden tip as a substrate and an ESI emitter was first proposed by Hu et al.63) The ionization scheme is similar to the paper spray ionization. It involves the application of a high voltage to the wooden substrate and to the mixture of analyte and spray solvent. Gas phase ions resulting from electrospray were obtained for the MS analysis. Wooden tips are naturally hard and porous; they can be easily made as pointed. The hydrophilic nature of wooden tips allows adhesion of different forms of samples on the tip surface. Unlike paper-based substrates, the narrow stick shape avoids rapid diffusion and evaporation of solvents, resulting in long signal durations. Wooden toothpicks have been frequently used as the substrates due to low cost and disposable features.64) This approach has been applied for various applications, such as analysis of toxic substance in food samples,65) drugs in urine and oral fluid,66) and metabolite fingerprinting.67) In addition, wooden tip-based ESI was demonstrated for rapid analysis of pharmaceutical samples in various forms, including tablets, granules, capsules, suspensions and oral liquids.68)

Polarization

In general, electrospray is performed by the application of high voltage on a capillary emitter, which is positioned in front of the mass spectrometer. This setup directs the liquid

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solution in the capillary to form a Taylor cone and charged droplets for MS analysis. Although this setup is quite popular, the direct contact of solvents and electrolytes with the metal capillary may occasionally give rise to undesired reactions.69,70) An alternative setup also allows the formation of a strong electric field between the emitter and MS inlet to induce electrospray from the sample solution. It involves the application of high voltage on the MS inlet and grounding on the ESI emitter. This method of ESI setup was patented by Mann et al.,71) which has been implemented in commercial mass spectrometers. However, many researchers worked towards complete avoidance of the contact of liquids with metal capillary. Stark et al. demonstrated an electrospray interface which is based on dielectric polarization induced by electric field.72,73) The electrospray is generated by applying high voltage at the outside wall of a capillary to transfer charges to the inside wall of the capillary by a displacement current. However, this method is dependent on alternation of potentials.74) Similarly, Zhang et al. used alternating current high voltages to perform a contactless spray, where the charge state of peptides was controllable.75) In addition, Girault et al. employed a constant high voltage power supply and an electrical circuit comprising two synchronized switches. This process is used to generate high voltage pulses for onsetting a spray from a liquid droplet placed over poly (methylmethacrylate) substrate.76) The principle of this contactless spray was proposed from a capacitive coupling effect. This technique termed as electrostatic-spray ionization has been used for analysis of adsorbed proteins,77) perfumes,78) and imaging applications.79) A nanoflow liquid chromatography (LC) pump is commonly used for maintaining the flow rate required for nanoelectrospray.80,81) Chen’s group employed an ultrasonicator available in almost all the chemical laboratories to develop a new ionization method, so called as ultrasonication-assisted spray ionization (UASI).82) A tapered capillary was used to drive solutions from a vial, positioned within an ultrasonicator to the MS orifice. The ultrasonication not only acts as the driving force to push the sample solution through the capillary to the outlet, but also as the energy source for the phase transition of analytes from liquid phase to gas-phase. Furthermore, there is no direct electric contact made on the tapered capillary outlet because polarization induced by the high electric field by the mass spectrometer also facilitated ionization process in UASI. UASI-MS is suitable for the monitoring of chemical reaction in real time because of its simple setup.83) It can be setup by simply exposing the reaction vial into an ultrasonic tank and placing a capillary inlet into the reaction vial. The tapered capillary outlet should be in proximity to a mass spectrometer. Once switching on the setup, ion species generated from the chemical reactions can be readily acquired by the mass spectrometer. Hsieh et al. demonstrated a continuous flow ion source named as contactless atmospheric pressure ionization (CAPI) without employing any additional pump system or driving mechanism. They used a short tapered capillary with 1 cm long as a sampling tool and spray emitter to realize the ionization.84) The movement of liquid through the capillary was solely based on capillary action. There is no any direct electric contact required to be made on the tapered capillary emitter thus the entire setup was extremely simple. The generation of electrospray from this contactless Page 4 of 11

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Fig. 4. Setup of the polarization induced ESI-MS. A cherry tomato was used as the sample loading substrate, where a sample droplet was deposited on the cherry tomato.88) Copyright (2015) John Wiley and Sons.

Fig. 3. Schematic representation of C-API MS, with sample delivery enabled by capillary action. A short tapered silica capillary [length, 1 cm; base o.d., 363 µm (or 323 µm without polyimide); tip o.d., 10 µm] was positioned vertically above an electrically isolated aluminum slide, with the outlet end placed orthogonal to the inlet of a metal capillary attached to the orifice of an ion trap mass spectrometer. The distance between the outlet of the silica capillary and the inlet of the metal capillary, attached to the MS orifice, was ∼1 mm. Before the measurements, the silica capillary was filled with a makeup solution [deionized water/acetonitrile (1 : 1, v⁄v)] by means of capillary action. The inlet end of the silica capillary was then dipped into a droplet of a sample (10 µL) put onto the surface of the aluminum slide. The inset provides an illustration of the hypothetical mechanism of C-API.84) Reproduced with permission from ref. 84. Copyright (2011) American Chemical Society.

setup is mainly due to polarization of emanating droplets from capillary tip (Fig. 3). Furthermore, the polarization is induced by the high electric field provided by the inlet of the mass spectrometer, occurring on the eluents from the capillary emitter. Due to this simplistic nature, C-API has been demonstrated to be suitable for monitoring online chemical reactions.85) The C-API ion source could also be used for multiple sample analysis by an automated movement of stage beneath the capillary.86) In a similar setup, Chang and co-workers used the capillary action to sample liquids present in plant parts and their subsequent ionization to study bioactive compounds. In addition, Chen’s group demonstrated the polarization effect on tiny droplets emerging out of microcapillaries,84) where the same effect should also be seen in larger droplets as well. Microdroplets from an automatic pipette at regular time intervals also gave rise to signal MS, specifically when positioned in proximity to the MS inlet.87) The droplet from the pipette experienced the polarization effect due to high voltage at MS inlet. The pressure difference at the same point allowed the droplet to enter MS orifice, leading to generation of gas-phase ions.87) Recently, another method, called as

polarization induced electrospray ionization (PI-ESI),88) was reported by the same group. Electrospray can be directly generated from a microliter-size droplets (4–10 µL) that is placed over a dielectric material (e.g., a cherry tomato), which is placed close to the inlet of a mass spectrometer, applied with a high voltage (Fig. 4). The obtained mass spectral profiles acquired using this method are similar to those obtained in conventional ESI mass spectra. Only a dielectric substrate for holding a sample droplet is required in PI-ESI,88) which is probably one of the simplest ESI variants that have been ever reported. One major advantage of PI-ESI is the avoidance of dead volume and adsorption surfaces which is typically seen in regular ESI.

Acoustics

It is well-known that acoustic waves can lead to atomization of liquid droplets by exponential growth of surface disturbances.89,90) Acoustic waves have been used as a simple and alternative means for nebulization and generation of electrospray from liquid samples in MS analysis.91–93) The first demonstration of using ultrasonic waves for generation of electrospray was conducted by Fenn and co-workers.91) An ultrasonic nebulizer was used to facilitate the nebulization of the liquid eluent from liquid chromatograph to form fine droplets.91) Later, Fedorov’s group tried to simplify the ultrasonic electrospray process by using micromachined ultrasonic atomizer. It is placed in 900 kHz–2.5 MHz range for droplet generation and a metal electrode in the fluid cavity for ionization.92) Ultrasound waves induce the formation of cavitations in liquid, resulting in bubble formation in the region where the pressure of liquid is below its vapor pressure. Due to collapsed bubbles, temperature and pressure are dramatically increased, resulting in the releasing of significant amount of energy for chemical reactions. Didenko and Suslick have demonstrated that radicals and ions are formed from cavitations.93) Chen’s group lately demonstrated that MS combined with a small metal chip equipped with a MHz-based ultrasonic transducer as an ion source can be used to the analysis of analytes with a wide mass range, such as amino acids, peptide, and small proteins.94) The mass spectral profiles of these analytes are similar to ESI mass spectra. Additionally, this ionization method can also be used to accelerate and monitor organic reactions in real time.94) Surface acoustic waves (SAWs) are Rayleigh waves with nanometer amplitude and propagate along the surface of Page 5 of 11

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a piezoelectric substrate at MHz order frequencies. SAWs have emerged as a powerful tool for actuation of microscale and nanoscale fluid and manipulation of bio-particles.95,96) Additionally, SAW nebulization (SAWN) has been recently used in the design for an open air ion source,97) which can be used to transfer non-volatile analytes directly from solution to the gas phase for MS analysis. The setup includes a surface acoustic wave device in the form of X-propagating lithium niobate wafer positioned beneath the capillary inlet of mass spectrometer. Multiply charged ions are generated in SAWN-MS. Due to the induced surface electric field that is associated with the SAW on the piezoelectric substrate, polarization of the liquid at the substrate surface and at the air/liquid interface occurs. Because of the electroelastic nature of surface acoustic waves, the droplets pinching-off from the substrate during atomization process are charged, from which gas phase ions are produced upon solvent evaporation.98) SAWN has been shown to be even softer than ESI.

INDIRECT ESI APPROACHES Desorption based ESI

The first desorption based ESI method, called as desorption electrospray ionization (DESI), was first reported by Cooks and co-workers in 2004.99) DESI allows in-situ desorption and ionization of analytes from the surface of samples under ambient conditions. The DESI process involves directing a solvent spray of charged micro-droplets from a pneumatically assisted sprayer towards the surface of interest under ambient conditions. The impact of spray desorbs the analyte into gas phase and subsequently ionization occurs.100) This feature of DESI-MS enables the analysis of specimen from native environment and under atmospheric pressure with minimal sample preparation. Various studies have been conducted to understand the mechanism behind DESI process.101–106) The most commonly described mechanism is droplet pick-up mechanism. That is, initial droplets derived from electrospray collide on a sample surface, where thin film of liquid is formed. This thin film of liquid dissolves the analytes present on the sample surface.

Fig. 5.

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Subsequent droplets keep hitting onto the surface and provide sufficient momentum to liberate the sample droplets containing analytes from the surface.104) Electrospray-like droplets are formed, which experience the cycles of solvent evaporation and Coulomb repulsion. Eventually, gas phase ions are formed for MS analysis. In addition, one also believes that gas phase ion/molecule reactions are also involved in ionization mechanisms.105,106) A phenomenon known as chemical sputtering may also attribute to the ion formation in DESI.102) Typical DESI-MS instrument consists of a solvent delivery line, a coaxial nozzle, nebulizing gas, high voltage power supply, and moving stages for controlling the position of samples with respect to the MS inlet (Fig. 5). Some of the important parameters which determine the ion signals in DESI-MS are incident angle of spray, collection angle of secondary droplets, distance between sprayer and surface, composition and flow rate of spray solvent, and type of surface.107) Over the years, DESI-MS has been applied in wide areas such as forensics,106,108) biology,109–111) chemistry,112–116) imaging,117,118) and cancer diagnostics.119–122) Another related technique known as desorption sonic-spray ionization (DeSSI)123) also uses the desorption phenomena like DESI. However, unlike DESI, a voltage free and sonic spray is used. This technique was later named as easy ambient sonic spray ionization (EASI).124) In EASI, the solution containing analyte is sprayed from a fused silica capillary with a supersonic nebulizing gas flow coaxial to the capillary. The substrate used in EASI is a cellulose dialysis membrane through which analytes are selectively permeated and then desorbed and ionized through DeSSI process.125,126) Nanospray desorption electrospray ionization (nanoDESI) was first reported by Roach et al. as an atmospheric pressure liquid extraction-ionization technique.127) In nanoDESI, a capillary tube applied with a high voltage and with continuous solvent infusion for generation of electrospray is used to mobilize compounds from sample surfaces. Solvated analytes are aspirated by a second capillary tube and subsequent ESI occurs at the outlet of the second capillary tube.127) In this technique, flow rate plays a crucial role as

Schematic representation of desorption electrospray ionization (DESI). Page 6 of 11

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the flow rate of the solvent from capillary has to be matched to the self-aspiration rate of the probe capillary. Due to the feature of nano-DESI to analyze analytes from surface, it has been applied extensively for imaging from a variety of samples.128–130) Furthermore, it has been applied in profiling sweat metabolites using hydrogel micropatches as sampling substrates.131,132)

Electrospray for post-ionization

The interaction of neutral species using electrospray as the post-ionization source for MS analysis has been investigated for some time.133–136) Post-ionization based on the use of electrospray is highly efficient for transferring charges from electrospray to neutral species. In post-ionization techniques, sampling and ionization occur in two distinct steps. The first step involves desorption of samples, and then the desorbed species are allowed to interact with an electrospray stream for ionization. Shiea and co-workers utilized ultrasonic nebulization by coupling an ultrasonic nebulizer to liberate samples from condensed-phase to gas phase and direct the desorbed species to an electrospray stream for post-ionization through fusion.136) Alternatively, to bring samples from condensed phase to gas phase was achieved by using a laser. Two types of lasers that have different ranges of wavelengths, for example, ultraviolet (UV) and infrared (IR), have been used to liberate samples from condensed phase to gas phase for post-ionization. The approach was first reported by Shiea et al.137) A UV laser was used to liberate condensed samples followed by post-ionization by electrospray named as electrospray assisted laser desorption/ionization (ELDI). The setup of ELDI consisted of a nitrogen laser (337 nm) operated at 20 µJ/pulse and a focused laser spot of 100×150 µm. The incident angle of the laser beam was set at 45°138) (Fig. 6). The desorbed species were ionized as a result of interaction with an electrospray steam. ELDI enabled the surface analysis of dried samples such as paintings, compact disc, drug tablets, and porcine brain tissue.139) In addition, Muddiman et al. reported a technique similar to ELDI, which is known as matrix assisted laser desorption electrospray ionization (MALDESI).140) Unlike ELDI, MALDESI requires lightabsorption matrices in the sample preparation. The presence of light-absorption matrices can facilitate ion formation during ionization. Vertes and co-workers alternatively used mid-IR laser for desorbing neutral species.141,142) The use of

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mid-IR laser allowed analysis of biological samples with sufficient water content. These approaches have been extensively applied for imaging applications of biological tissues.141,142) Another related technique known as laser electrospray mass spectrometry (LEMS) uses a femtosecond laser for desorption and electrospray as post-ionization.143) LEMS has been applied for analysis of a wide range of samples such as explosives,144) smokeless powders,145) pharmaceuticals,146) and biological macromolecules.147) Although laser desorption has been used as a means to generate gas-phase ions from condensed phase, these approaches demonstrated that ionization efficiency of desorbed species can be further improved by post-ionization with electrospray.

CONCLUSION ESI-MS is one of the most versatile analytical tools available for various analytes. This review has attempted to provide a perspective on new modifications and recent developments of ESI. In a brief period of time, a large number of ESI derived variants with a myriad of practical applications have been developed. These modifications have gained popularity due to the fact that it has simplified the notion of ESI-MS analysis. The increasing availability of robust mass spectrometers has allowed chemists to explore various methods based on the use of electrospray for MS analysis. These electrospray-based ionization methods have minimized the requirement of sample pre-treatments for various analysis. Although few practical limitations still exist, they can be overcome with further developments. Although these newly developed ionization methods cannot completely replace traditional ESI-MS, they have certainly opened up new possibilities in the field of analytical chemistry and vision in MS research. It is highly possible that new applications with the use and the improvement of these newly developed ionization techniques can be further explored.

Acknowledgements We thank the Ministry of Science and Technology of Taiwan for financial support (MOST102-2113-M-009-019-MY3 and MOST 105-2113-M-009-022-MY3) of this work. AKM acknowledges National Chiao Tung University for providing him NCTU International Student Scholarship.

REFERENCES 1)

2) 3) 4)

Fig. 6. Detailed illustration of the ELDI MS setup. (A) Sampling skimmer of a mass analyzer; (B) nitrogen laser beam; (C) electrospray capillary; (D) sample plate; (E) focusing lens; (F) reflecting lens; (G) syringe pump. Reprinted with permission from ref. 138. Copyright (2006) American Chemical Society.

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