Sheathless Electrospray Ionization Research Articles

Compact automated capillary electrophoresis instrument for coupling with mass spectrometry by using sheathless electrospray ionization

The low-cost set-up is based on the flexible microfluidic breadboard approach. To avoid the use of electrically driven devices at the high voltage injection end of the capillary pneumatically actuated pumps and valves are employed. It is coupled to the mass spectrometer with a sheathless electrospray obtained in an electrodeless interface configuration. This simplification is achieved by carrying out the analysis in two distinct steps, firstly the separation without the formation of an electrospray, and secondly detection in which the electrospray is created while pumping out the content of the capillary. The performance of the instrument was tested by analysing three benzalkonium ions and the four pesticides pyrifenox, pirimicarb, cyprodinil and pyrimethanil. Detection limits between 0.36 and 0.76 µM and peak reproducibilities between 5.2% and 6.6% (50 µM standards) were obtained for the latter.

Effect of the flow profile on separation efficiency in pressure-assisted reversed-polarity capillary zone electrophoresis of anions: Simulation and experimental evaluation.

Capillary electrophoresis connected to electrospray ionization mass spectrometry is a promising combination to analyze complex biological samples. The use of sheathless electrospray ionization interfaces, such as a porous nanoelectrospray capillary emitter, requires the application of forward flow (either by pressure or electroosmosis) to maintain the electrospray process. The analysis of solute molecules with strong negative charges (e.g., aminopyrenetrisulfonate labeled glycans) necessitates a reversed-polarity capillary electrophoresis separation mode, in which case the electroosmotic flow is counter current, thus pressure assistance is necessary. In this study, we compared the effect of forced convection with and without counter electroosmotic flow on the resulting separation efficiency in capillary electrophoresis based on flow profile simulations by computational fluid dynamics technique and by actual experiments. The efficiencies of the detected peaks were calculated from the resulting electropherograms and found approximately 950 000 plates/m for electrophoresis with counter electroosmotic flow, 20000 plates/m with pressure only (such as would be in open tubular liquid chromatography), and 480 000 plates/m for electrophoresis with simultaneous counter electroosmotic flow and forward pressure assistance, which validates the simulation data.

Hyphenation of Production-Scale Free-Flow Electrophoresis to Electrospray Ionization Mass Spectrometry Using a Highly Conductive Background Electrolyte.

In this technical note, we demonstrate the hyphenation of production-scale free-flow electrophoresis (FFE) and sheathless electrospray ionization mass spectrometry (ESI-MS). In contrast to previous hyphenation approaches, we used a highly conductive background electrolyte (BGE) required for production-scale FFE. We found that this kind of BGE as well as a production-scale setup leads to significant electric interference between FFE and MS. This interference prevents steady-state FFE operation. We examine this interference in detail and discuss possible solutions to this issue. We demonstrate that the straightforward grounding of the transfer line removes the influence of ESI-MS on FFE, but creates a current leak from the ESI interface, which adversely affects the ESI spray. Furthermore, we show that only the electrical disconnection of the ESI probe from the FFE-MS transfer line suppresses this undesirable current. In order to facilitate the electrical disconnection we used a low conductivity, silica-based ESI probe with withdrawn inner capillary. This approach allowed the interference-free hyphenation of production-scale FFE (using a highly conductive BGE) with ESI-MS.

Hyphenation of capillary high-performance ion-exchange chromatography with mass spectrometry using sheath-flow electrospray ionization.

Mass spectrometry (MS) is an attractive method for extending capillary-size ion chromatography (cHPIC) to create a valuable technique for speciation analysis. For hyphenation, the aqueous effluent of cHPIC has to be transformed into a volatile mixture for MS while preserving analytical concentrations as well as peak shapes during transfer from cHPIC to MS. Finally, the approach should technically be flexible and easy-to-use. A combination of cHPIC and sheath-flow electrospray ionization (ESI)-MS offers to solve all these challenges. cHPIC/sheath-flow-ESI-TOFMS was used in this study for the speciation analysis of various arsenic model compounds. These model compounds were analyzed with different hyphenation setups and configurations of cHPIC/MS and their respective assets and drawbacks were examined and discussed. The parameters (flow rate and composition of sheath liquid) of sheath-flow ESI and their influence on the performance of the spray and the sensitivity of the detector were investigated and compared with those of sheathless ESI. Using an injection valve to couple cHPIC and MS was found to be the best method for hyphenation, since it constitutes a flexible and dead-volume-free approach. The investigation of sheath-flow ESI revealed that the flow rate of the sheath liquid has to resemble the flow rate of the IC effluent to ensure a stable spray and that a composition of 2-propanol/water/ammonia at 50:50:0.2 (v/v/v) suits most applications without unilaterally promoting the sensitivity for either organic or inorganic compounds. The optimized setup and conditions were successfully applied to the analysis of a mixture of important arsenic species and used to determine limits of detection of organic and inorganic arsenic species (3.7 µg L(-1) elemental arsenic). A method for cHPIC/sheath-flow-ESI-MS was developed. The method was shown to be a valuable tool for speciation and trace analysis. It features no dead volume, fast transfer from IC to MS, only minimal peak-widening, high reproducibility, and the ability to fine-tune the ESI spray for higher sensitivity and stability by adjusting the composition of the sheath-liquid.

Amino acid analysis of dried blood spots for diagnosis of phenylketonuria using capillary electrophoresis-mass spectrometry equipped with a sheathless electrospray ionization interface

We describe a capillary electrophoresis-mass spectrometry (CE-MS) method for newborn screening of a representative amino acid metabolic disease, namely, phenylketonuria (PKU). Underivatized phenylalanine and tyrosine in a dried blood spot (DBS) were simultaneously determined by CE-MS equipped with an ionophore membrane-packed sheathless electrospray ionization interface, which was developed by our group. The method was optimized for rapid determination of the underivatized amino acids, phenylalanine and tyrosine extracted from a DBS. Under the optimized conditions, the limit of detection of phenylalanine and tyrosine (signal-to-noise ratio, 3) was 0.03 and 0.07 mg/L in DBS, respectively, with a CE run time of less than 3 min. For repeated runs of a sample, coefficients of variation (CVs) for migration time were less than 3.7%, whereas CVs for the area ratio under the curve were 2.1 and 2.9% for 20 consecutive runs of 49.5 mg/kg Phe and 36.2 mg/kg Tyr, respectively. However, the relative standard deviations of intra- and interday assays for DBS samples were <6.2 and <5.8%, respectively, which were substantially due to sample extraction from DBS. The analytical method was applied to real clinical samples of Korean neonates, and results were compared with those of conventional methods for PKU diagnosis, which required reference analytical methods such as isotope dilution CE-MS or high-performance liquid chromatography-mass spectrometry for quality assurance of the conventional kit-based assays. The distinct advantages of high sensitivity and extremely low sample volume, as well as a simple, easy, and economic sample pretreatment, were demonstrated for the proposed method.

Low-Flow Sheathless Capillary Electrophoresis–Mass Spectrometry for Sensitive Glycoform Profiling of Intact Pharmaceutical Proteins

Capillary electrophoresis coupled to time-of-flight mass spectrometry (CE-TOF-MS) via a porous tip sheathless electrospray ionization (ESI) interface was studied for the characterization of pharmaceutical glycoproteins. To achieve optimal glycoform separation, background electrolytes of low pH were used in conjunction with a capillary with a neutral coating exhibiting near-zero electroosmotic flow. Crucial interfacing parameters, like ESI voltage and ESI tip-to-end plate distance, were optimized for very low flow rates (∼5 nL/min) in order to attain maximum sensitivity and stable performance. Under optimal conditions, the sheathless CE-MS interface provided significantly increased ionization efficiencies for intact proteins and decreased ionization suppression leading to detection limits in the picomolar-range. Analysis of a sample of recombinant human interferon-β allowed the assignment of at least 18 glycoforms, plus a variety of deamidation, succinimide, and oxidation products, representing a considerable improvement over sheath-liquid CE-MS. The sheathless CE-MS system also proved highly suitable for the glycoprofiling of recombinant human erythropoietin, revealing 74 glycoforms in a 60-min run. In addition, oxidation and acetylation products were detected, overall resulting in assignment of more than 250 different isoforms. Semiquantitative glycoprofiles could be derived for both pharmaceutical proteins, with estimated glycoform concentrations analyzed ranging from 0.35 to 950 nM. These profiles may be very useful for quality control of biopharmaceuticals and their biosimilars.

Capillary electrophoresis mass spectrometry with sheathless electrospray ionization for high sensitivity analysis of underivatized amino acids

A high durability sheathless electrospray ionization interface of CE-MS is applied for the sensitive analysis of underivatized amino acids. The sheathless interface was realized using an ionophore membrane-packed electro-conduction channel. The interface functioned well with a volatile alkaline background electrolyte (BGE) and uncoated fused-silica capillaries for CE-MS analysis of underivatized amino acids. High electroosmotic flow with alkaline BGE facilitated high separation efficiency (>100,000 theoretical plates) and short analysis time (<15 min). Both the short-term stability and long-term durability are particularly suited for routine applications. Using electrokinetic injection and the multiple reaction monitoring (MRM) mode with a triple-quadrupole analyzer, high sensitivity was achieved, which yielded detection limits of 0.05-0.81 μM. For the quantitation of underivatized amino acids, quantification precisions (RSDs) for intra- and inter-day analyses were less than 3%. Recoveries from serum were 96.3-101.8% for isotope dilution mass spectrometry (IDMS). When compared with HPLC-IDMS for human serum samples, highly agreeable (96.9-102.0%) results were obtained with the proposed CE-IDMS method.

Characterization of drug-lysozyme conjugates by sheathless capillary electrophoresis–time-of-flight mass spectrometry

Drug-protein conjugates have been widely used for the cell-specific targeting of drugs to cells that can bind and internalize the proteinaceous carrier. For renal drug targeting, lysozyme (LZM) can be used as an effective carrier that accumulates in proximal tubular cells. We used capillary electrophoresis–time-of-flight mass spectrometry (CE–TOF-MS) for the characterization of different drug-LZM conjugates. A recently developed prototype porous tip sprayer was employed for sheathless electrospray ionization (ESI) CE–MS interfacing. In order to prevent adsorption of LZM conjugates to the capillary wall, a positively charged polyethylenimine capillary coating was used in combination with a low-pH background electrolyte. Drug-LZM products had been prepared by first coupling BOC- l-methionine hydroxysuccinimide ester (BOCmet) to lysine residues of LZM followed by conjugation with the kinase inhibitors LY364947, erlotinib, or Y27632 via a platinum(II)-based linker. CE–TOF-MS of each preparation showed narrow symmetrical peaks for the various reaction products demonstrating that drug-LZM conjugates remained stable during the CE analysis and subsequent ESI. Components observed in the drug-LZM products were assigned based on their relative migration times and on molecular mass as obtained by TOF-MS. The TOF-MS data obtained for the individual components revealed that the preparations contained LZM carrying one or two drug molecules, next to unmodified and BOCmet-modified LZM. Based on relative peak areas (assuming an equimolar response for each component) a quantitative conjugate profile could be derived for every preparation leading to drug loading values of 0.4–0.6 mol drug per mole protein.

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification.

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off-line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure-assisted flow. Unlike first-dimensional ion-exchange LC fractionation, where there is a large degree of overlap, the CZE-MS results show less than 15% overlap between neighboring RPLC fractions.

Comparison between different sheathless electrospray emitter configurations regarding the performance of nanoscale liquid chromatography–time-of-flight mass spectrometry analysis

Four different sheathless electrospray ionization (ESI) configurations were investigated for a nano liquid chromatography (LC) system. The studied configurations were: a column with an integrated emitter, with the ESI potential applied before or after the column, and a column with separate emitter, with the ESI voltage applied at a union before the emitter or at the emitter tip. The results indicates that the efficiency of the LC system is rather independent of the configuration when using 95 μm i.d. columns, acetic mobile phase and standard peptides as a sample. Introduction of post column dead volume seems not to be a critical issue at least with flow rates down to 600 nl/min.

Sheathless electrospray ionization directly from a capillary monolith for fast liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry

Sheathless electrospray ionization directly from a capillary monolith for fast liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry

On-column sample enrichment for capillary electrophoresis sheathless electrospray ionization mass spectrometry: evaluation for peptide analysis and protein identification.

Although several designs have been advanced for coupling sample enrichment devices to a sheathless electrospray ionization-mass spectrometry (MS) interface on a capillary electrophoresis (CE) column, most of these approaches suffer from difficulties in fabrication, and the CE separation efficiency is degraded as a result of the presence of coupling sleeves. We have developed a design that offers significant improvements in terms of ease of fabrication, durability, and maintenance of the integrity of the CE-separated analyte zones. Capillaries with different inside and outside diameters were evaluated to optimize the performance of the CE-MS system, resulting in a mass limit of detection of 500 amol for tandem MS analysis of a standard peptide using a 20-microm-i.d. capillary. The improved design incorporates an efficient method to preconcentrate a sample directly within the CE capillary followed by its electrophoretic separation and detection using a true zero dead-volume sheathless CE-MS interface. Testing of this novel CE-MS system showed its ability to characterize proteomic samples such as protein digests, in-gel-digested proteins, and hydrophobic peptides as well as to quantitate ICAT-labeled peptides.

Development of a Poly(dimethylsiloxane) Interface for On-Line Capillary Column Liquid Chromatography−Capillary Electrophoresis Coupled to Sheathless Electrospray Ionization Time-of-Flight Mass Spectrometry

An interface in elastomeric poly(dimethylsiloxane) (PDMS) for on-line orthogonal coupling of packed capillary liquid chromatography (LC) (i.d. = 0.2 mm) with capillary electrophoresis (CE) in combination with sheathless electrospray ionization (ESI) time-of-flight mass spectrometric (TOFMS) detection is presented. The new interface has a two-level design, which in combination with a continuous CE electrolyte flow through the interface provides integrity of the LC effluent and the CE separation until an injection is desired. The transparent and flexible PDMS material was found to have a number of advantages when combined with fused silica column technology, including ease to follow the process and ease to exchange columns. By combining conventional microscale systems of LC, CE, and ESI-MS, respectively, the time scales of the individual dimensions were harmonized for optimal peak capacity per unit time. The performance of the LC-CE-TOFMS system was evaluated using peptides as model substances. A S/N of about 330 was achieved for leucine-enkephaline from a 0.5 microL LC injection of 25 microg/mL peptide standard.

A conductive polymeric material used for nanospray needle and low-flow sheathless electrospray ionization applications.

A conductive polypropylene/graphite mixture is used for the production of polymeric nanospray needle emitters and as a coating on fused-silica capillaries that are used for sheathless electrospray ionization (ESI). The described production of these polymeric nanospray needle emitters and sheathless ESI contacts is exceptionally easy and at a very low cost. The described polymeric nanospray emitters have shown excellent features regarding their chemical inertness and spray performance. The long-term stability of the nanospray needles exceeds 24 h of continuous use. Furthermore, the resistance to electrical discharges, which is one of the factors that often limits the lifetime of metal coated tips, has proven to be outstanding. A voltage of up to 5 kV could be applied without loss of spray performance. The use of polypropylene emitters offers a number of desirable features, as compared to silica based emitters. Among these features are mechanical flexibility and simplified regeneration of the nanospray needle. Continuous nanospray of peptides and proteins in conjunction with orthogonal time-of-flight mass spectrometry are shown with signal relative standard deviations of 5%. In addition, the polypropylene/graphite mixture has also been applied as the conductive contact for sheathless ESI in fast capillary electrophoresis separations.

Evaluations of the stability of sheathless electrospray ionization mass spectrometry emitters using electrochemical techniques.

The processes that cause the failure of sheathless electrospray ionization (ESI) emitters, based on different kinds of gold coatings on fused-silica capillaries, are described and explained. The methods chosen for this study include electrochemical methods, ICPMS analysis of the electrolytes used, SEM studies, and electrospray experiments. Generally, the failure occurs by loss of the conductive coating. It is shown that emitters with sputter-coated gold lose their coatings because of mechanical stress caused by the gas evolution accompanying water oxidation or reduction. Emitters with gold coatings on top of adhesion layers of chromium and nickel alloy withstand this mechanical stress and have excellent durability when operating as cathodes. When operating as anodes, the adhesion layer is electrochemically dissolved through the gold film, and the gold film then flakes off. It is shown that the conductive coating behaves as a cathode even in the positive electrospray mode when the magnitude of a superimposed reductive electrophoretic current exceeds that of the oxidative electrospray current. Fairy-dust coatings developed in our laboratory (see Barnidge, D. R.; etal.Anal. Chem. 1999, 71, 4115-4118,) bygluing gold dust onto the emitter, are unaffected by the mechanical stress due to gas evolution. When oxidized, the fairy-dust coatings show an increased surface roughness and decreased conductivities due to the formation of gold oxide. The resistance of this oxide layer is however negligible in comparison with that of the gas phase in ESI. Furthermore, since no flaking and only negligible electrochemical etching of gold was found, practically unlimited emitter lifetimes may be achieved with fairy-dust coatings.