Multi-charged oligonucleotide ion formation in sonic spray ionization.

An oligonucleotide tends to release hydrogen atoms from a phosphoric acid group and to form negative ions that can be detected by mass spectrometry. Usually, with a solution-spray based ionization technique, the negative ions are present in different charge states. Ion formation for the nucleotide is quite complicated and is easily influenced by matrix and other constituents in a sample solution, as well as by the operating parameters for a mass spectrometer. In this work, we studied oligonucleotide ion formation by using an ion trap mass spectrometer combined with a sonic spray ionization (SSI) source. An oligonucleotide with 20 bases was measured. Effects from contaminants and parameters affecting the ion production, such as a high voltage applied to the ionization source and sample solution-flow rate, were investigated. Our results showed that an ion with about one charge for every three bases was most abundant. However, the signal intensity and the mass spectrum pattern were sensitive to the matrix and operating parameters. One of the reasons for such sensitivity is that there are various ion states for an oligonucleotide. Any change in the matrix or an operating parameter may shift the balances between the ion states. Adding Tris, or (hydroxymethyl)aminomethane, enhanced the signal intensity of the oligonucleotide and promoted formation of the oligonucleotide ion with higher charges, while adding acetic acid favored the ions with lower charges, compared with that obtained in the medium without adding Tris and acetic acid. The effects on charged droplets and chemical enhancement were investigated. The mechanism for oligonucleotide ion formation is discussed.     

A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry

A brief account of the mechanisms by which ions in solution are converted to ions in the gas phase is given on the basis of information available in the literature and the four companion articles on electrospray mass spectrometry (ESMS) in this issue. The following stages/phenomena are described: (a) production of the charged droplets at the ES capillary tip; (b) evolution of the charged droplets due to solvent evaporation and droplet fission caused by Coulombic repulsion of the charges on the droplets; production of the gas phase ion from very small charged droplets by the charge residue model (CRM) or the ion evaporation method (IEM); (c) dependence of the sensitivity in ESMS on the chemical nature of the analyte and its concentration as well as on the concentration of other electrolytes that are present in the solution; qualitative predictions on the sensitivity of the analyte based on the surface activity of the analyte ions; (d) relationship between ions produced in the gas phase and original ions present in the solution; and (e) globular proteins. Much of the information presented in (a)-(e) has been available for some time in the literature. However some significant advances are relatively recent. Recent results by de la Mora and co-workers, including their contribution in this Special Feature, provide very strong evidence that small ions (in distinction from macroions such as bio-macroions) are produced by IEM. On the other hand, macroions and particularly the polyprotonated globular proteins are produced by CRM. Also noteworthy is the development of an equation by Enke with which the observed relative ion signal intensities of the gas-phase ions produced can be predicted on the basis of the ion concentration in solution over a wide concentration range. The recognition that the sensitivity of organic analyte ions can be qualitatively predicted on the basis of the hydrophilicity or hydrophobicity of the part of the molecule that is not part of the charged (ionic) group and affects the surface activity of the ionic species is also noteworthy and a very useful relatively recent development.     

Capillary electrophoresis-electrospray mass spectra of the herbicides paraquat and diquat

The positive ion electrospray mass spectra of the quaternary ammonium salt herbicides paraquat and diquat are examined by on-line separation with capillary electrophoresis (CE) and by direct infusion of the analytes. The analytes are separated by CE in 7–10 min at pH 3.9 in 50% methanol-water by using several different separation buffer electrolytes. The capillary electrophoresis-electrospray ionization (CE-ES) mass spectra of paraquat and diquat consist primarily of doubly charged molecular ions, singly charged molecular ions, and singly charged deprotonated ions. The direct infusion spectra consist primarily of doubly charged molecular ions and singly charged deprotonated ions. The relative abundances of the doubly charged and deprotonated ions depend strongly on the presence or absence of ammonium ion in the CE separation buffer or the direct infusion solution. A deprotonation mechanism is proposed in which the free base ammonia is the deprotonating agent in the desolvating charged droplets or in the gas phase. The analytical potential of the CE-ES electrospray approach for environmental analyses is evaluated in terms of the precision of replicate injections, linear concentration range, and estimated detection limit.     

Ultrasonication-assisted spray ionization mass spectrometry for the analysis of biomolecules in solution

In this paper, we describe a novel technique—ultrasonication-assisted spray ionization (UASI)—for the generation of singly charged and multiply charged gas-phase ions of biomolecules (e.g., amino acids, peptides, and proteins) from solution; this method employs a low-frequency ultrasonicator (ca. 40 kHz) in place of the high electric field required for electrospray ionization. When a capillary inlet is immersed into a sample solution within a vial subjected to ultrasonication, the solution is continually directed to the capillary outlet as a result of ultrasonication-assisted capillary action; an ultrasonic spray of the sample solution is emitted at the outlet of the tapered capillary, leading to the ready generation of gas-phase ions. Using an ion trap mass spectrometer, we found that singly charged amino acid and multiply charged peptides/proteins ions were generated through this single-step operation, which is both straightforward and extremely simple to perform. The setup is uncomplicated: only a low-frequency ultrasonicator and a tapered capillary are required to perform UASI. The mass spectra of the multiply charged peptides and proteins obtained from sample solutions subjected to UASI resemble those observed in ESI mass spectra.     

A cylindrical capacitor ionization source: droplet generation and controlled charge reduction for mass spectrometry

A cylindrical capacitor ionization source was used in conjunction with corona discharge charge reduction for generation of singly charged ions for mass spectrometric analysis. The source consists of a fused-silica capillary threaded with a platinum wire and placed inside a stainless steel tube. Application of an electric potential to the wire results in the production of a linear stream of charged droplets when an aqueous solution is pumped through the capillary. Subsequent solvent evaporation yields ions, providing a continuous ion source for mass spectrometry. Passage of the ions through a corona discharge charge reduction chamber permits reduction of the charge state to predominantly singly charged species, facilitating analysis of DNA and protein mixtures. The change from production of multiply charged ions to production of singly charged ions is extremely simple, requiring only modulation of the voltage applied to the corona discharge electrode. A simple technique for construction of the ionization source is reported.     

Ionization and transmission efficiency in an electrospray ionization—mass spectrometry interface

The ionization and transmission efficiencies of an electrospray ionization (ESI) interface were investigated to advance the understanding of how these factors affect mass spectrometry (MS) sensitivity. In addition, the effects of the ES emitter distance to the inlet, solution flow rate, and inlet temperature were characterized. Quantitative measurements of ES current loss throughout the ESI interface were accomplished by electrically isolating the front surface of the interface from the inner wall of the heated inlet capillary, enabling losses on the two surfaces to be distinguished. In addition, the ES current lost to the front surface of the ESI interface was spatially profiled with a linear array of 340-microm-diameter electrodes placed adjacent to the inlet capillary entrance. Current transmitted as gas-phase ions was differentiated from charged droplets and solvent clusters by measuring sensitivity with a single quadrupole mass spectrometer. The study revealed a large sampling efficiency into the inlet capillary (>90% at an emitter distance of 1 mm), a global rather than a local gas dynamic effect on the shape of the ES plume resulting from the gas flow conductance limit of the inlet capillary, a large (>80%) loss of analyte ions after transmission through the inlet arising from incomplete desolvation at a solution flow rate of 1.0 microL/min, and a decrease in analyte ions peak intensity at lower temperatures, despite a large increase in ES current transmission efficiency.     

Minimizing analyte electrolysis in an electrospray emitter

The electrospray (ES) ion source is a controlled-current electrolytic flow cell. Electrolytic reactions in the ES emitter capillary are continually ongoing to sustain the production of charged droplets and ultimately gas-phase ions from this device. Under certain circumstances, the analytes under study may be directly involved in these electrolytic processes. It is demonstrated that a simple means to minimize analyte electrolysis is to exchange the normal metal emitter capillary of commercial ES sources with one made of fused silica. This change is shown to provide an ES mass spectrometric system of similar performance in terms of gas-phase ion signal generated for non-electroactive analytes and also assures minimal oxidation of electroactive analytes even at low (2.0 microl x min(-1)) solution flow-rates and high (millimolar) solution electrolyte concentrations.     

Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods

This paper reports a detailed study of the internal energy distribution of ions formed by four electrospray ionization (ESI)-related ionization methods, with particular emphasis on electrosonic spray ionization (ESSI). Substituted benzylpyridinium ions were used as thermometer ions to probe the internal energy distribution. The influence of different instrumental parameters was studied. Cone and skimmer voltages as well as the collision energy were found to strongly affect the ion internal energy distribution, whereas the distance between the emitter and the inlet of the mass spectrometer, the nebulizing gas pressure or the flow rate showed no influence. The internal energy distribution obtained with an ESSI source was compared with those obtained for electrospray (ESI), nanoelectrospray (nanoESI) and sonic spray ionization (SSI) on the same mass spectrometer with the same instrumental parameters. No clear differences were observed. As the charge residue model is the only ion formation mechanism possible for SSI, we conclude that benzylpyridinium ions are formed by the pathway suggested by this model.     

Particle discriminator interface for nanoflow ESI-MS

An atmosphere to vacuum interface was designed to exploit the different mobility and momentum characteristics of ions, and charged and neutral particles in electrospray ionization-mass spectrometry. The purpose of this device is to transmit with high efficiency the ions created at atmospheric pressure into the mass analyzer and to deflect the large charged and neutral particles prior to entrance into the vacuum system, thereby maintaining system cleanliness and stability. This interface is particularly suitable for low flow rate electrospray ionization-mass spectrometry where the close proximity of the electrospray emitters to the vacuum entrance, and near total consumption of the entire spray, leads to the production of large quantities of non-desolvated droplets and large charged and neutral particles. The improvement involves the application of potential gradients to a particle discriminator space located between the gas restricting ion entrance orifice of the mass spectrometer and the exit of a heated laminar flow chamber to divert large particles from the gas conductance limiting orifice. A counter-current flow of drying gas is used to deflect neutral particles and solvent vapor. Two stages of desolvation are achieved with the combined effects of the curtain gas and heated laminar flow chamber. This enhances the efficiency of desolvation and ion production, and stabilizes the resulting ion current under a wide variety of solvent compositions. In addition, this system eliminates the problems associated with the boiling of solution in nanospray tips when operated in close proximity to a heated mass spectrometer inlet. The particle discriminator interface gives approximately a 2-fold improvement in ion count rates, and a 3-fold improvement in stability (as measured by the signal relative standard deviation).     

Redox buffering in an electrospray ion source using a copper capillary emitter.

An electrospray ion source used in electrospray mass spectrometry is a two-electrode, controlled-current electrochemical flow cell. Electrochemical reactions at the emitter electrode (oxidation and reduction in positive and negative ion modes respectively) provide the excess charge necessary for the quasi-continuous production of charged droplets and ultimately gas-phase ions with this device. We demonstrate here that a copper capillary emitter, in place of the more commonly used stainless-steel capillary emitter, can be utilized as a redox buffer in positive ion mode. Anodic corrosion of the copper capillary during normal operation liberates copper ions to solution and in so doing maintains the interfacial potential at this electrode near the equilibrium potential for the copper corrosion process [E degrees = 0.34 V versus standard hydrogen electrode (SHE)]. Fixing the interfacial potential at the emitter electrode provides control over the electrochemical reactions that take place at this electrode. It is shown that the oxidation of N-phenyl-1,4-phenylenediamine to N-phenyl-1,4-phenylenediimine (E(p/2) = 0.48 V versus SHE) can be completely avoided using the copper emitter, whereas this analyte is completely oxidized with a stainless-steel capillary emitter under the same conditions. Moreover, using N-phenyl-1,4-phenylenediimine, we demonstrate that reduction reactions can occur at the copper emitter electrode in positive ion mode. Emitter corrosion, in addition to redox buffering, provides a convenient means to introduce metal ions into solution for analytical use in electrospray mass spectrometry.     

Direct analysis of oligosaccharides and alpha hydroxy acids in fruits using electrosonic spray ionization mass spectrometry

Electrosonic spray ionization (ESSI) is a derivative technique of electrospray ionization (ESI) for mass spectrometry (MS) in which droplets are charged in the course of sonic spray. In this study, we applied ESSI MS to direct analysis of oligosaccharides and alpha hydroxy acids (AHAs) in fruits. The components were extracted from fruit fleshes by a feasible method prior to ESSI MS analysis, but the fruit juices were analyzed without further pretreatment. The results demonstrate that mainly alkali metal adducts of oligosaccharides are favorably produced in positive ion mode, while deprotonated AHAs and oligosaccharides are produced in negative ion mode. Compared with mass spectra obtained using electrospray droplet impact/secondary ion mass spectrometry (EDI/SIMS), mass spectra using ESSI make the identification of oligosaccharides more straightforward in positive ion mode than in negative ion mode.     

Study of the desorption/ionization mechanism in electrospray droplet impact secondary ion mass spectrometry

Electrospray droplet impact (EDI) secondary ion mass spectrometry (SIMS) is a desorption/ionization technique for mass spectrometry in which highly charged water clusters produced from an atmospheric-pressure electrospray are accelerated in vacuum by several kV and impact on the sample deposited on the metal substrate. The abundances of the secondary ions for C(60) and amino acids are measured as a function of the acceleration voltage of the primary charged water droplets. Two desorption/ionization mechanisms are suggested in the EDI ionization processes: low-energy and high-energy regimes. In the low-energy regime, the excess charges in the primary droplets play a role in the formation of secondary ions. In the high-energy regime, samples are ionized by the supersonic collision of the primary droplets with the sample. The yield of secondary ions increases by about three orders of magnitude with increase in the acceleration voltage of the primary droplets from 1.75 kV to 10 kV.     

Electrode‐assisted desorption electrospray ionization mass spectrometry

A new ion source has been developed for rapid, noncontact analysis of materials at ambient conditions. The method provides desorption of analytes under ambient conditions directly from different surfaces with little or no sample preparation. The new method, termed electrode‐assisted desorption electrospray ionization (EADESI), is on the basis of the ionization of molecules on different surfaces by highly charged droplets produced on a sharp‐edged high voltage tip, and ions produced are introduced into the mass spectrometer through a capillary. The EADESI technique can be applied to various samples including amino acids, peptides, proteins, drugs and human fluids such as urine and blood. EADESI is promising for routine analyses in different fields such as forensic, environmental and material sciences. EADESI interface can be fit to a conventional ion‐trap mass spectrometer. It can be used for various types of samples with a broad mass range. EADESI can also provide real‐time analysis which is very valuable for biomedical applications. Copyright © 2010 John Wiley & Sons, Ltd.     

Desorption sonic spray ionization for (high) voltage-free ambient mass spectrometry

Sonic spray ionization is shown to create a supersonic cloud of charged droplets able to promote efficient desorption and ionization of drugs directly from the surfaces of commercial drug tablets at ambient conditions. Compared with desorption electrospray ionization (DESI), desorption sonic spray ionization (DeSSI) is advantageous since it uses neither heating nor high voltages at the spray capillary. DeSSI therefore provides a more friendly environment in which to perform ambient mass spectrometry (MS). DeSSI-MS is herein evaluated for the analysis of drug tablets, and found to be, in general, as sensitive as DESI-MS. The (high) voltage-free DeSSI method provides, however, cleaner mass spectra with less abundant solvent cluster ions and with enough abundant analyte signal for tandem mass spectrometry (MS/MS). These features may therefore facilitate the DeSSI-MS detection of low molar mass components or impurities, or both. The higher-velocity supersonic DeSSI spray also facilitates matrix penetration thus providing more homogenous sampling and longer lasting ion signals.     

Metal-ligand solution equilibria studied by electrospray ionization mass spectrometry: effect of instrumental parameters

Electrospray ionization mass spectrometry (ESI-MS) is being increasingly employed in the study of metal-ligand equilibria in aqueous solution. In the present work, the ESI-MS spectral changes due to different settings of the following instrumental parameters are analyzed: the solution flow rate (F(S)), the nebulizer gas flow rate (F(G)), the sprayer potential (E), and the temperature of the entrance capillary (T). Twenty-eight spectra were obtained for each of six samples containing aluminum(III) and 2,3-dihydroxypyridine at various pH, in the absence or in the presence of a buffer and of sodium ions. Among the considered instrumental parameters, T produced the largest effects on the ionic intensities. F(S) and F(G) affected the ESI-MS spectra to a lower extent than T. In the investigated conditions E had the weakest effects on the spectra.The correlations observed between the ionic intensities and these instrumental parameters were interpreted considering the presence of three kinds of perturbations occurring in the ESI-MS ion source: formation of some dimers in the droplets, different transfer efficiencies from the droplets to the gas phase for different complexes (according to their surface activity), and subsequent partial thermal decomposition of the dimers and of one of the monomeric complexes in the gas phase. Our results show that the evaluation of the effects produced in the ESI-MS spectra by a change of instrumental parameters can allow to identify the perturbations occurring when metal-ligand solutions are studied by ESI-MS.     

Desorption ionization by charge exchange (DICE) for sample analysis under ambient conditions by mass spectrometry

An ambient pressure ionization technique for mass spectrometric analysis of substances present on solid surfaces was developed. A nebulized spray containing molecular ions of a solvent such as toluene can be generated by passing the solvent through a stainless steel capillary held at a high voltage. When the stream of charged droplets produced in this way is directed onto a solid surface, the analytes present on the surface are desorbed and ionized by a charge exchange process. This technique was shown to desorb and generate positively charged molecular ions from compounds that are not readily ionized by some other ambient methods, under positive-ion generation mode. For example, intense signals representing radical cations of 1,4-hydroquinone, limonene, thymol, and several other compounds were observed when the analytes were deposited on a metal surface and exposed to a toluene spray nebulized from the metal capillary maintained at a potential of about +5 kV. In contrast, when the same samples were exposed to a spray of water/methanol/formic acid under customary DESI-like (positive-ion mode) conditions, no peaks representing the analytes were observed.     

Numerical Modeling of Ion Transport in an ESI-MS System

Gas and ion transport in the capillary-skimmer subatmospheric interface of a mass spectrometer, which is typically utilized to separate unevaporated micro-droplets from ions, was studied numerically using a two-step approach spanning multiple gas dynamic regimes. The gas flow in the heated capillary and in the interface was determined by solving numerically the Navier-Stokes equation. The capillary-to-skimmer gas/ion flow was modeled through the solution of the full Boltzmann equation with a force term. The force term, together with calculated aerodynamic drag, determined the ion motion in the gap between the capillary and skimmer. Three-dimensional modeling of the impact of the voltage applied to the Einzel lens on the transmission of doubly charged peptide ions through the skimmer orifice was compared with experimental data obtained in the companion study. Good agreement between measured and computed signals was observed. The numerical results indicate that as many as 75% of the ions that exit from the capillary are lost on the conical surface of the skimmer or capillary outer surface because of the electrostatic force and plume divergence.

Signal enhancement in desorption nanoelectrospray ionization by custom‐made inlet with pressure regulation

The efficiency of desorption/ionization becomes more critical as the sampled surface area decreases. Desorption electrospray and desorption nanoelectrospray belong to ambient ionizations and enable direct surface analysis including mass spectrometric imaging. Lateral resolution in tens of micrometers was demonstrated for desorption nanoelectrospray previously, but sensitivity of the surface scan can be an issue. For desorption electrospray, the drag force in the source is driven by the flow of used gases and vacuum suction. Ion signal intensity can be improved by controlling the nebulizing gas flow rate or auxiliary pumping of a closed compartment in front of the mass spectrometer inlet. Because nanoelectrospray generates charged droplets without the assistance of a nebulizing gas, only vacuum suction drives the gas flow. In this study, the effect of pressure drop between the atmospheric and evacuated region of a mass spectrometer on the ion signal intensity was investigated for desorption nanoelectrospray. A modification of the commercial inlet was designed. An auxiliary pump was directly connected to an inner compartment of the modified mass spectrometer inlet through a needle valve that enabled the regulation of the reduced pressure. Adjustment of the pressure drop significantly increased signal intensity (more than one order of magnitude in some cases). To a lesser extent, the temperature of a heated capillary (an integral part of the inlet) also influenced the signal intensity. The applicability of desorption nanoelectrospray equipped with pressure regulation was demonstrated by the analysis of synthetic cathinones or a pill of paracetamol. Because pressure in the inlet depends on the diameters of orifices and the power of vacuum systems of mass spectrometers, the effect of the pressure regulation can be different for different instruments. Nevertheless, the presented results confirmed the importance of pressure drop‐driven transport for desorption nanoelectrospray efficiency and can encourage its new applications.     

Laserspray ionization (LSI) ion mobility spectrometry (IMS) mass spectrometry

A simple device is described for desolvation of highly charged matrix/analyte clusters produced by laser ablation leading to multiply charged ions that are analyzed by ion mobility spectrometry-mass spectrometry. Thus, for example, highly charged ions of ubiquitin and lysozyme are cleanly separated in the gas phase according to size and mass (shape and molecular weight) as well as charge using Tri-Wave ion mobility technology coupled to mass spectrometry. This contribution confirms the mechanistic argument that desolvation is necessary to produce multiply charged matrix-assisted laser desorption/ionization (MALDI) ions and points to how these ions can be routinely formed on any atmospheric pressure mass spectrometer.     

Investigation on the role of pneumatic aspects in electrospray, desorption electrospray surface ionization and surface activated chemical ionization

This review reports the results of some studies carried out by us on the role of pneumatic aspects in electrospray and desorption electrospray surface ionization, with the aim to propose some relevant aspects of the mechanisms involved in these ionization methods. Electrospray ion sources, with the exception of the nano- electrospray source, operate with the concurrent action of a strong electrical field and a supplementary coaxial gas flow. The electrical field is responsible for electrospraying of the analyte solution but the use of a coaxial gas flow leads to a significant increase of the analyte signal and allows the use of higher solution flows. However, by employing capillary voltages much lower than those necessary to activate the electrospray phenomenon, analyte ions are still observed and this indicates that different mechanisms must be operative for ion production. Under these conditions, ion generation could take place from the neutral pneumatically sprayed droplet by field-induced droplet ionization. Also in the case of desorption electrospray ionization (DESI), and without any voltage on the spraying capillary as well as on the surface of interest, ions of analytes present on the surface become detectable and this shows that desorption/ionization of analytes occurs by neutral droplets impinging the surface. Consequently, the pneumatic effect of the impinging droplets plays a relevant role, and for these reasons the method has been called pneumatic assisted desorption (PAD). Some analogies existing between PAD and surface activated chemical ionization (SACI), based on the insertion of a metallic surface inside an atmospheric pressure chemical ionization source operating without corona discharge, are discussed.