A pulsed triple ionization source for sequential ion/ion reactions in an electrodynamic ion trap

A pulsed triple ionization source, using a common atmosphere/vacuum interface and ion path, has been developed to generate different types of ions for sequential ion/ion reaction experiments in a linear ion trap-based tandem mass spectrometer. The triple ionization source typically consists of a nano-electrospray emitter for analyte formation and two other emitters, an electrospray emitter and an atmospheric pressure chemical ionization emitter or a second nano-electrospray emitter for formation of the two different reagent ions. The three emitters are positioned in a parallel fashion close to the sampling orifice of the tandem mass spectrometer. The potentials applied to each emitter are sequentially pulsed so that desired ions are generated separately in time and space. Sequential ion/ion reactions take place after analyte ions of interest and different set of reagent ions are sequentially injected into a linear ion trap, where axial trapping is effected by applying an auxiliary radio frequency voltage to the end lenses. The pulsed triple ionization source allows independent optimization of each emitter and can be readily coupled to any atmospheric pressure ionization interface with no need for instrument modifications, provided the potentials required to transmit the ion polarity of interest can be synchronized with the emitter potentials. Several sequential ion/ion reactions examples are demonstrated to illustrate the analytical usefulness of the triple ionization source in the study of gas-phase ion/ion chemistry.     

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.     

Electrolytic deposition of metals on to the high-voltage contact in an electrospray emitter: implications for gas-phase ion formation

The electrospray ion source is an electrolytic flow cell. Electrolytic reactions in the electrospray emitter maintain the production of charged droplets by this ion source that contain an excess of ions of one polarity. These redox reactions necessarily change the composition of the solution that initially enters the emitter. As a result, the ions ultimately observed in the gas phase by electrospray mass spectrometry (ESMS) may be substantially influenced by both the nature and extent of these electrochemical reactions. It is demonstrated in this paper that Ag(+), Cu(2+) and Hg(2+) ions in solution can be electrolytically reduced and deposited as the respective metals on to the surface of the high-voltage contact in the electrospray emitter in negative ion mode electrospray. The deposited metals are shown to be liberated from the surface by switching the electrospray high-voltage polarity to operate in the positive ion mode. The deposited metals are oxidized in positive ion mode, releasing the metal ions back into solution where they are detected in the electrospray mass spectrum. In a semi-quantitative analysis, it was found that up to 50% of the Ag(+) in a 2.5 microM solution was deposited on the high-voltage contact of the emitter as the solution flowed through the emitter. Deposition of Cu(2+) and Hg(2+) was less efficient. These data illustrate that in the analysis of metals by ESMS, one must be aware that both the concentration and form of the metals may be altered by electrochemical processes in the emitter. Hence reduction or oxidation of metals in the electrospray emitter, which may remove ions from solution, or change metal valence, would be expected to impact both quantitative metal determinations and metal speciation attempts using ESMS.     

Pulsed dual electrospray ionization for In/In reactions

A pulsed dual electrospray ionization source has been developed to generate positive and negative ions for subsequent ion/ion reaction experiments. The two sprayers, typically a nano-electrospray emitter for analytes and an electrospray emitter for reagents, are positioned in a parallel fashion close to the sampling orifice of a triple quadrupole/linear ion trap tandem mass spectrometer (Sciex Q TRAP). The potentials applied to each sprayer are alternately pulsed so that ions of opposite polarity are generated separately in time. Ion/ion reactions take place after ions of each polarity are sequentially injected into a high-pressure linear ion trap, where axial trapping is effected by applying an auxiliary radio frequency voltage to the end lenses. The pulsed dual electrospray source allows optimization of each sprayer and can be readily coupled to any spray interface with no need for instrument modifications, provided the potentials required to transmit the ion polarity of interest can be alternated in synchrony with the emitter potentials. Ion/ion reaction examples such as charge reduction of multiply charged protein ions, charge inversion of peptides ions, and protein-protein complex formation are given to illustrate capabilities of the pulsed dual electrospray source in the study of gas-phase ion/ion chemistry.     

Minimizing analyte electrolysis in electrospray ionization mass spectrometry using a redox buffer coated emitter electrode

An emitter electrode with an electroactive poly(pyrrole) (PPy) polymer film coating was constructed for use in electrospray ionization mass spectrometry (ESI‐MS). The PPy film acted as a surface‐attached redox buffer limiting the interfacial potential of the emitter electrode. While extensive oxidation of selected analytes (reserpine and amodiaquine) was observed in positive ion mode ESI using a bare metal (gold) emitter electrode, the oxidation was suppressed for these same analytes when using the PPy‐coated electrode. A semi‐quantitative relationship between the rate of oxidation observed and the interfacial potential of the emitter electrode was shown. The redox buffer capacity, and therefore the lifetime of the redox buffering effect, correlated with the oxidation potential of the analyte and with the magnitude of the film charge capacity. Online reduction of the PPy polymer layer using negative ion mode ESI between analyte injections was shown to successfully restore the redox buffering capacity of the polymer film to its initial state. Published in 2010 by John Wiley & Sons, Ltd.     

Electron capture dissociation of singly and multiply phosphorylated peptides

Analysis of phosphotyrosine and phosphoserine containing peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissociation (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissociation (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z(.)-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z(.) fragment ion products was observed in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatography combined with nano-electrospray FTMS/ECD facilitated phosphopeptide analysis and amino acid sequencing from crude proteolytic peptide mixtures.     

萃取纳升喷雾-离子迁移谱法现场快速筛查化妆品中8种禁用抗生素

采用萃取纳升喷雾结合离子迁移谱技术,建立了化妆品中8种禁用抗生素的现场快速筛查方法。对萃取纳升喷雾毛细管拉制条件、萃取纳升喷雾离子化条件、离子迁移谱检测条件等进行了详细考察和优化。在优化的实验条件下,8种禁用抗生素的方法检出限为20 mg/kg,离子迁移谱分析时间小于20 ms,单个样品全部检测时间不超过30 s。对于筛检出的疑似阳性样品,进一步采用超高效液相色谱-串联质谱进行确证。该方法流程简便、快捷高效,为化妆品中违禁组分的现场快速筛查提供了较为广阔的应用前景。     

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.     

Efficient analyte oxidation in an electrospray ion source using a porous flow-through electrode emitter

This article describes the components, operation, and use of a porous flow-through electrode emitter in an electrospray ion source. This emitter electrode geometry provided enhanced mass transport to the electrode surface to exploit the inherent electrochemistry of the electrospray process for efficient analyte oxidation at flow rates up to 800 microL/min. An upstream current loop in the electrospray source circuit, formed by a grounded contact to solution upstream of the emitter electrode, was utilized to increase the magnitude of the total current at the emitter electrode to overcome current limits to efficient oxidation. The resistance in this upstream current loop was altered to control the current and "dial-in" the extent of analyte oxidation, and thus, the abundance and nature of the oxidized analyte ions observed in the mass spectrum. The oxidation of reserpine to form a variety of products by multiple electron transfer reactions and oxidation of the ferroceneboronate derivative of pinacol to form the ES active radical cation were used to study and to illustrate the performance of this new emitter electrode design. Flow injection, continuous infusion, and on-line HPLC experiments were performed.     

Characterization of solution-phase and gas-phase reactions in on-line electrochemistry-thermospray tandem mass spectrometry

Electrochemistry was used on-line with high-performance liquid chromatography-thermospray tandem mass spectrometry to provide insight into the solution-phase decomposition reactions of electrochemically generated oxidation products. Products formed during electrooxidation were monitored as the electrode potential was varied. The solution reactions which follow the initial electron transfer at the electrode are affected by the vaporizer tip temperature of the thermospray probe and the composition of the thermospray buffer. Either hydrolysis or ammonolysis reactions of the initial electrochemical oxidation products can occur with pH 7 ammonium acetate buffer. Both the electrochemically generated and the synthesized disulfide of 6-thiopurine decompose under thermospray conditions to produce 6-thiopurine and purine-6-sulfinate. Solution-phase studies indicate that nucleophilic and electrophilic substitution reactions with purine-6-sulfinate result in the formation of purine, adenine, and hypoxanthine. Products were identified and characterized by tandem mass spectrometry. This work shows the first example of high-performance liquid chromatography used on-line with electrochemistry to separate stable oxidation products prior to analysis by thermospray tandem mass spectrometry. In addition, solution-phase and gas-phase studies with methylamine show that the site of the nucleophilic and electrophilic reactions is probably inside the thermospray probe. Most importantly, these results also show that the on-line combination of electrochemistry with thermospray tandem mass spectrometry provides valuable information about redox and associated chemical reactions of biological molecules such as the structures of intermediates or products as well as providing insight into reaction pathways.     

Numerical simulation of Monte Carlo ion transport at atmospheric pressure within improved air amplifier geometry

A computational fluid dynamics (CFD) software package ANSYS Fluent was employed for simulation of ion transport at atmospheric pressure between a nano-electrospray ionization (nano-ESI) emitter and the mass spectrometer (MS) sampling inlet tube inside an improved air amplifier device incorporating a radiofrequency ion funnel. The flow field, electric field and the ion trajectory calculations were carried out in separate steps. Parallelized user-defined functions were written to accommodate the additional static and transient electric fields and the elastic ion-gas collisions with the Monte Carlo hard-sphere simulation abilities within Fluent’s environment. The ion transmission efficiency from a nano-ESI emitter to the MS sampling inlet was evaluated for different air amplifier and ion funnel operating conditions by tracking 250 sample reserpine ions. Results show that the high velocity gas stream and the external electric field cause a rapid acceleration of the ion beam and its dispersion along the centreline of the air amplifier which leads to reduction of the space-charge effect and the beam divergence. The radiofrequency potential applied to the ion funnel contributed to additional ion focusing.     

Redox transformations in desorption electrospray ionization

Redox changes occur in some circumstances when organic compounds are analyzed by desorption electrospray ionization mass spectrometry (DESI-MS). However, these processes are limited in scope and the data presented here suggest that there are only limited analogies between the redox behavior in DESI and the well-known solution-phase electrochemical processes in standard electrospray ionization (ESI). Positive and negative ion modes were both investigated and there is a striking asymmetry between the incidence of oxidation and of reduction. Although in negative ion mode DESI experiments, some aromatic compounds were ionized as odd-electron anion radicals, examples of full reduction were not found. By contrast, oxidation in the form of oxygen atom addition (or multiple oxygen atom additions) was observed for several different analytes. These oxidation reactions point to chemically rather than electrochemically controlled processes. Data is presented which suggests that oxidation is predominantly caused by reaction with discharge-created gas-phase radicals. The fact that common reducing agents and known antioxidants such as ascorbic acid are not modified, while a saturated organic acid like stearic acid is oxidized in DESI, indicates that the usual electrochemical redox reactions are not significant but that redox chemistry can be induced under special experimental conditions.     

An open design microfabricated nib-like nanoelectrospray emitter tip on a conducting silicon substrate for the application of the ionization voltage

This paper describes a novel emitter tip having the shape of a nib and based on an open structure for nano-electrospray ionization mass spectrometry (nanoESI-MS). The nib structure is fabricated with standard lithography techniques using SU-8, an epoxy-based negative photoresist. The tip is comprised of a reservoir, a capillary slot and a point-like feature, and is fabricated on a silicon wafer. We present here a novel scheme for interfacing such nib tips to MS by applying the ionization voltage directly onto the semi-conductor support. The silicon support is in direct contact with the liquid to be analyzed at the reservoir and microchannel level, thus allowing easy use in ESI-MS. This scheme is especially advantageous for automated analysis as the manual step of positioning a metallic wire into the reservoir is avoided. In addition, the analysis performance was enhanced compared with the former scheme, as demonstrated by the tests of standard peptides (gramicidin S, Glu-fibrinopeptide B). The limit of detection was determined to be lower than 10(-2) microM. Due to their enhanced performance, these microfabricated sources might be of great interest for analysis requiring very high sensitivity, such as proteomics analysis using nanoESI-MS.     

Resin-packed nanoelectrospray in combination with video and mass spectrometry for the direct and real-time molecular analysis of mast cells

A nano-electrospray ionization (nanoESI) emitter for analysis of a biological solution was developed by packing a nanoESI needle with two types of resins for desalting and preconcentration of target molecules. Determination of secreted histamine and serotonin molecules in cell culture buffers was demonstrated using 5-methyltryptamine as internal standard. The results showed good linearity of target signals in the concentration range from 0.25 to 50.0 ng/mL of histamine or serotonin. These molecules were monitored to be secreted by A23187 (calcium ionophore) stimulant in rat peritoneal mast cells. Using a combination of a video-microscope and a mass spectrometer, we could visualize exocytotic moments and analyze secreted molecules by mass spectrometry simultaneously. Time-dependent release of histamine and serotonin from activated mast cells showed that significant production of these molecules occurred and reached a maximal level at 15 min for serotonin and at 30 min for histamine, respectively. These results showed that this method allows the direct and timely analysis of secreted molecules in biological responses.     

Development of rapid methodologies for the isolation and quantitation of drug metabolites by differential mobility spectrometry – mass spectrometry

Clinical and forensic toxicology laboratories are inundated with thousands of samples requiring lengthy chromatographic separations prior to mass spectrometry. Here, we employ differential mobility spectrometry (DMS) interfaced to nano-electrospray ionization-mass spectrometry to provide a rapid ion filtration technique for the separation of ions in gas phase media prior to mass spectral analysis on a DMS-integrated AB SCIEX API 3000 triple-quadrupole mass spectrometer. DMS is efficient at the rapid separation of ions under ambient conditions and provides many advantages when used as an ion filtration technique in tandem with mass spectrometry (MS) and MS/MS. Our studies evaluated DMS-MS/MS as a rapid, quantitative platform for the analysis of drug metabolites isolated from urine samples. In targeted applications, five metabolites of common drugs of abuse were effectively and rapidly separated using isopropanol and ethyl acetate as transport gas modifiers, eliminating the gas chromatography or liquid chromatography-based separations commonly employed in clinical and forensic toxicology laboratories. Calibration curves were prepared for the selected drug metabolites utilizing deuterated internal standards for quantitative purposes. The feasibility of separating and quantitating drug metabolites in a rapid fashion was evaluated by compensation voltage stepping followed by multiple reaction monitoring (MRM) detection. Rapid profiling of clinical and forensic toxicology samples could help to address an urgent need within the scientific community by developing high-throughput analytical methodologies, which could reduce significant case backlogs present within these laboratories.     

亚微米电喷雾喷针在非变性质谱分析中的研究进展

非变性质谱技术已成为表征蛋白质结构的重要工具之一。与传统的电喷雾喷针(Electrospray emitter,ESI emitter)相比,亚微米电喷雾喷针具有改变离子电荷态分布和降低盐离子加合等多种特性,可在生理环境下直接解析蛋白质的结构。该文综述了亚微米电喷雾喷针的特性及其在非变性质谱分析中的应用,并对其未来的发展趋势进行了展望。     

Loss of charged versus neutral heme from gaseous holomyoglobin ions.

The dissociation of holomyoglobin ions ranging in charge state from +10 to +2 has been studied using collisional activation in a quadrupole ion trap. Collisional activation times and amplitudes were varied to investigate the effects of these variables on dissociation of the heme group from the holoprotein. The onset of neutral heme loss occurs at a lower activation amplitude than loss of charged heme. For solutions of ferri-myoglobin, charged heme loss was prominent for +10 to +4 holomyoglobin ions, while neutral heme loss product was found to be dominant for charge states +3 and +2. For any given charge state, activation of holomyoglobin ions from a solution containing primarily ferro-myoglobin yielded significantly more abundant neutral heme loss products than was observed for activation of ions from solutions containing primarily ferri-myoglobin. The relative concentrations of the two oxidation states were shown to be affected by redox chemistry within the nano-electrospray emitter used in this work. Results from a double activation experiment revealed that the precursor ions of a given charge state contained a mixture of two populations, with ferro-myoglobin giving rise to neutral heme loss upon dissociation and ferri-myoglobin yielding charged heme. No evidence for electron transfer upon collisional activation of ferri-myoglobin ions was observed. Furthermore, little or no evidence for electron transfer associated with ion/ion reactions with anions derived from perfluoro-1,3-dimethylcyclohexane was observed. Definitive results could not be drawn for the lowest precursor ion charge states (+3 and +2) due to low dissociation efficiencies.     

Field-Desorption Mass Spectra of Fern Phloroglucinols Containing Three to Six Ring Constituents

It has been previously shown that the structural analysis of the title compounds by electron-impact ionization (EI)mass spectrometry is complicated by extensive, if not complete, thermal rottlerone-type rearrangement and/or fragmentation reactions. The use of a ‘softer’ ionization technique such as field desorption (FD) is, therefore, required in order that these compounds may be characterized in terms of their molecular size and composition. In this report, the FD mass spectra of typical representatives of this class of compounds are described along with the attempted suppression of undesirable thermal reactions by the optimization of the emitter temperatures.     

常压敞开式离子化-离子迁移谱法快速筛查玩具中14种致癌致敏染料

采用常压敞开式离子化结合离子迁移谱技术,研究建立了蜡笔、水贴纸、橡皮泥等玩具样品中14种致癌致敏染料的快速筛查方法。无需繁琐的样品前处理过程,玩具样品经纸喷雾或萃取纳升喷雾,将上样、萃取、电离等步骤集成为一步实现,并在16 ms内完成了离子迁移谱分析检测。同时还对疑似阳性样品建立了超高效液相色谱-串联质谱的确证方法。14种致癌致敏染料的检出限为0.5~2 mg/kg。该方法流程便捷、快速高效,适用于玩具样品的现场快速筛查。     

Matrix effects in negative-ion field desorption mass spectrometry

The negative ion mode of field desorption mass spectrometry is matrix dependent. The matrix is used to facilitate the desolvation of ions at field strengths below the onset of field induced electron detachment. The role of the matrix in the desolvation mechanism for negative ions is briefly described and the main properties of a matrix solution, such as viscosity, solubility for complex salts and acids, and adhesion to the emitter surface, on the ion formation are discussed. The application of matrix mixtures with strong adhesion to the emitter surface even at elevated temperatures and high field strengths are considered to be important for the further improvement of this ionization method.