Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated fused-silica capillary

A simple procedure was developed for preparing a carbon-coated fused-silica capillary for use in sheathless capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The tapered capillary tip was smeared with a marker pen before coating with carbon using a soft pencil. The layer from the ink of the marker pen was critical to the preparation of the carbon-coated capillary. The fabrication of a carbon-coated fused-silica capillary tip requires less than 1 min. The stability of this carbon-coated fused-silica capillary is examined, and its utility in on-line sheathless CE/ESI-MS is demonstrated with the separation of berberine, coptisine, and palmatine chlorides. Although the carbon-coated fused-silica capillary tip is not as rugged as a gold-coated capillary, it is durable enough for sheathless CE/ESI-MS applications. Moreover, it is easy to refurbish the column once the performance of the tip is degraded.     

Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated tapered fused-silica capillary with a beveled edge.

A tapered capillary tip containing a beveled edge was developed for use in sheathless capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The optimal flow rate of a 75-microm-i.d., 90-microm-o.d. beveled tapered capillary tip was similar to a conventional flat tapered tip with a 25-microm orifice. Using a mixture of coptisine, berberine, and palmatine chloride, the sheathless CE/ ESI-MS sensitivity of a beveled 75 microm tapered tip capillary was found to be similar to a 25 microm flat tip. Although both tips offer similar CE/ESI-MS sensitivity, the beveled tapered capillary tip is more rugged and durable than a conventional 25-microm tapered capillary because of the larger outside diameter and inside diameter. To make electrical contact, the capillary tip was smeared with paint marker followed by the application of a carbon coating using a graphite pencil. Using this refined carbon-coating procedure, the capillary tip can be operated with aprotic solvents.     

Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol-water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 mumol/L and for acidic and low molecular weight amino acids were less than 6.0 mumol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.     

A colloidal graphite-coated emitter for sheathless capillary electrophoresis/nanoelectrospray ionization mass spectrometry

A colloidal graphite-coated emitter is introduced for sheathless capillary electrophoresis/nanoelectrospray ionization time-of-flight mass spectrometry (CE/ESI-TOFMS). The conductive coating can be produced by brushing the capillary tip to construct a fine layer of 2-propanol-based colloidal graphite. The fabrication involves a single step and requires less than 2 min. Full cure properties develop in approximately 2 h at room temperature and then the tip is ready for use. The coated capillary tip is applied as a sheathless electrospray emitter. The emitter has proven to bear stable electrospray and excellent performance for 50 microm i.d. x 360 microm o.d. and 20 microm i.d. x 360 microm o.d. capillaries within the flow rate of 80-500 nL/min; continuous electrospray can last for over 200 h in positive mode. Baseline separation and structure elucidation of two clinically interesting basic drugs, risperidone and 9-hydroxyrisperidone, are achieved by coupling pressure-assisted CE to ESI-TOFMS using the described sheathless electrospray emitter with a bare fused-silica capillary at pH 6.7. It is found that the signal intensity of m/z in sheathless CE/ESI-TOFMS at pH 6.7 is approximately 50 times higher than that at pH 9.0 for the two analytes, although the electroosmotic flow (EOF) at pH 9.0 provides sufficient flow rate (approximately 150 nL/min) to maintain electrospray.     

Pressure-assisted capillary electrophoresis electrospray ionization mass spectrometry for analysis of multivalent anions

We describe a method, based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS), that allows the simultaneous and quantitative analysis of multivalent anions, such as citrate isomers, nucleotides, nicotinamide-adenine dinucleotides, and flavin adenine dinucleotide, and coenzyme A (CoA) compounds. Key to the analysis was using a noncharged polymer, poly(dimethylsiloxane), coated to the inner surface of the capillary to prevent anionic species from adsorbing onto the capillary wall. It was also necessary to drive a constant liquid flow toward the MS by applying air pressure to the inlet capillary during electrophoresis to maintain a conductive liquid junction between the capillary and the electrospray needle. Although theoretical plates were inferior to those obtained by CE/ESI-MS using a cationic polymer-coated capillary, the PACE/ESI-MS method improved reproducibility and sensitivity of these anions. Eighteen anions were separated by PACE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. The relative standard deviations (n = 6) of the method were better than 0.6% for migration times and between 1.4% and 6.2% for peak areas. The detection limits for these species were between 0.4 and 3.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL), that is, mass detection limits calculated in the range from 12 to 110 fmol at a signal-to-noise ratio of 3. The utility of the method was demonstrated by analysis of citrate isomers, nucleotides, dinucleotides, and CoA compounds extracted from Bacillus subtilis cells.     

A low-makeup beveled tip capillary electrophoresis /electrospray ionization mass spectrometry interface for micellar electrokinetic chromatography and nonvolatile buffer capillary electrophoresis

A robust interface has been developed for interfacing micellar electrokinetic chromatography (MEKC) and nonvolatile buffer capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS). The interface consists of two parallel capillaries for separation (50 microm i.d. x 155 microm o.d.) and makeup (50 microm i.d. x 155 microm o.d.) housed within a larger capillary (530 microm i.d. x 690 microm o.d.). The capillaries terminate in a single tapered tip having a beveled edge. The use of a tapered beveled edge results in a greater tip orifice diameter (75 microm) than in a previous design from our laboratory (25 microm) that used a flat tip. While maintaining a similar optimum flow rate and consequently similar sample dilution, a 75-microm beveled emitter is more rugged than a 25-microm flat tip. Furthermore, the incorporation of a sheath liquid capillary allows the compositions of the final spray solution to be controlled. The application of this novel CE/ESI-MS interface was demonstrated for MEKC using mixtures of triazines (positive ion mode) and phenols (negative ion mode). The ability to perform CE/ESI-MS using a nonvolatile buffer was demonstrated by the analysis of gangliosides with a buffer consisting of 40 mM borate and 20 mM alpha-cyclodextrin.     

Fully integrated glass microfluidic device for performing high-efficiency capillary electrophoresis and electrospray ionization mass spectrometry

A microfabricated device has been developed in which electrospray ionization is performed directly from the corner of a rectangular glass microchip. The device allows highly efficient electrokinetically driven separations to be coupled directly to a mass spectrometer (MS) without the use of external pressure sources or the insertion of capillary spray tips. An electrokinetic-based hydraulic pump is integrated on the chip that directs eluting materials to the monolithically integrated spray tip. A positively charged surface coating, PolyE-323, is used to prevent surface interactions with peptides and proteins and to reverse the electroosmotic flow in the separation channel. The device has been used to perform microchip CE-MS analysis of peptides and proteins with efficiencies over 200,000 theoretical plates (1,000,000 plates/m). The sensitivity and stability of the microfabricated ESI source were found to be comparable to that of commercial pulled fused-silica capillary nanospray sources.     

Analysis of underivatized amino acids and their D/L-enantiomers by sheathless capillary electrophoresis/electrospray ionization mass spectrometry

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of underivatized amino acids and the separation of their D/L-enantiomers. Under full-scan mode, all standard protein amino acids were separated and detected at low-femtomole levels using a 130-cm-long, 20-microm-i.d., 150-microm-o.d. underivatized fused-silica capillary with 1 M formic acid as the background electrolyte. The CE/ESI-MS technique was also applied to the separation of L-arginine from L-canavanine (a close analogue of arginine where the terminal methylene linked to the guanidine group of arginine is replaced by an oxygen atom) in a complex mixture containing all standard protein amino acids. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the identification of two metabolic diseases (PKU and tyrosinemia) through blood analysis with minimal sample preparation. In addition, the on-line separation of 11 underivatized L-amino acids from their D-enantiomers was achieved by using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the background electrolyte.     

Frontal analysis capillary electrophoresis hyphenated to electrospray ionization mass spectrometry for the characterization of the antithrombin/heparin pentasaccharide complex

The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. A new strategy based on the hyphenation of frontal analysis capillary electrophoresis (FACE) with electrospray ionization mass spectrometry (ESIMS) is reported for the characterization of protein/carbohydrate complexes. While most of the previously reported CE-MS experiments were performed using capillary electrophoresis in zone format, we report for the first time CE-MS experiments in which CE was performed in frontal analysis (FACE-MS). We showed that the frontal mode offered a better sensitivity than zone mode and was well suited for the CE-MS coupling. This FACE-MS coupling was applied to the analysis of the complex between antithrombin and the sulfated pentasaccharide reproducing the antithrombin-binding sequence in heparin. The mixture of coincubated antithrombin and heparin pentasaccharide was continuously injected into the capillary, and the electrophoretic separation of the free and bound forms of the protein was achieved. The intact noncovalent complex antithrombin/heparin pentasaccharide was detected on-line by ESIMS in positive ionization mode and in nondenaturing sheath liquid conditions. The complex stoichiometry was determined from the mass measurement of the complex. In addition, the characterization of the sulfated pentasaccharide ligand dissociated from the complex was performed in negative ionization mode using a denaturing sheath liquid, allowing the determination of its molecular mass and sulfation features. This FACE-ESIMS strategy opens the way to ligand fishing experiments performed on heterogeneous carbohydrate mixtures and subsequent characterization of specifically bound carbohydrates.     

Simultaneous determination of anionic intermediates for Bacillus subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry

A method for simultaneous determination of anionic metabolites based on capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry is described. To prevent current drop by the system, electroosmotic flow (EOF) reversal by using a cationic polymer-coated capillary was indispensable. A mixture containing 32 standards including carboxylic acids, phosphorylated carboxylic acids, phosphorylated saccharides, nucleotides, and nicotinamide and flavin adenine coenzymes of glycolysis and the tricarboxylic acid cycle pathways were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. Key to the analysis was EOF reversal using a cationic polymer-coated capillary and an electrolyte system consisting of 50 mM ammonium acetate, pH 9.0. The relative standard deviations of the method were better than 0.4% for migration times and between 0.9% and 5.4% for peak areas. The concentration detection limits for these metabolites were between 0.3 and 6.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL); i.e., mass detection limits ranged from 9 to 200 fmol, at a signal-to-noise ratio of 3. This method was applied to the comprehensive analysis of metabolic intermediates extracted from Bacillus subtilis, and 27 anionic metabolites could be directly detected and quantified.     

A sheathless nanoflow electrospray interface for on-line capillary electrophoresis mass spectrometry

A novel, rugged capillary electrophoresis-electrospray ionization (CE-ESI) interface where the separation column, an electrical porous junction, and the spray tip are integrated on a single piece of a fused-silica capillary is described. ESI is accomplished by applying an electrical potential through an easily prepared porous junction across a 3-4-mm length of fused silica. A stable electrospray is produced at nanoflow rates generated in the capillary by electrophoretic and electroosmotic forces. The interface is particularly well suited for the detection of low-femtomole levels of proteins and peptides. The ruggedness of this interface was evident by the continuous operation of the same column for over a 2-week period with no detectable deterioration in separation or electrospray performance. The new interface was used for the LC-ESI-MS separation and analysis of peptides and proteins. Injection of 25 fmol of [Glu1]-fibrinopeptide B using the new device produced a CE-ESI-MS electropherogram with a signal-to-noise ratio of over 100 for this peptide.     

Characterization of the microdialysis junction interface for capillary electrophoresis/microelectrospray ionization mass spectrometry

A capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS) interface, based on an electric circuit across a microdialysis membrane surrounding a short capillary segment closely connected to the separation capillary terminus, is demonstrated to be sensitive, efficient, and rugged. A microspray type ionization emitter produces a stable electrospray at the low flow rates provided by CE and thus avoids both the need for a makeup liquid flow provided by liquid junction or sheath flow interfaces and the subsequent dilution and reduction in sensitivity. Reproducibility studies and comparisons with CE/UV and the CE/sheath flow interface with ESI-MS are presented. Additionally, postrun acidification via the microdialysis junction interface is demonstrated and shown to be capable of denaturing the holomyoglobin protein noncovalent complex while maintaining separation efficiency.     

Identifying specific small-molecule interactions using electrospray ionization mass spectrometry

A simple method for establishing whether complexes composed of small molecules detected by electrospray ionization mass spectrometry (ES-MS) originate from specific interactions in solution or nonspecific binding during the ES process is described. The technique, referred to as the nonspecific probe method, exploits the tendency of small molecules to bind nonspecifically to macromolecules during the ES process to establish the presence of specific noncovalent interactions. To implement the method, a macromolecule probe (P(NS)), which does not bind specifically to any of the components present in solution, is added prior to ES-MS analysis. The existence of specific small-molecule complexes is determined from an analysis of the measured distributions of the small molecules bound nonspecifically to P(NS). The principal assumption on which this methodology is based is that nonspecific binding of small molecules and their complexes to P(NS) during ES is a statistical (random) process. A mathematical framework for establishing the presence of specific heterocomplexes is presented. The reliability of the method for distinguishing specific from nonspecific small-molecule interactions is illustrated for peptide-antibiotic and metal ion-ligand interactions in water.     

Tissue imaging using nanospray desorption electrospray ionization mass spectrometry

Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here, we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 μm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.     

Array of chemically etched fused-silica emitters for improving the sensitivity and quantitation of electrospray ionization mass spectrometry

An array of emitters has been developed for increasing the sensitivity of electrospray ionization mass spectrometry (ESI-MS). The linear array consists of 19 chemically etched fused-silica capillaries arranged with 500 microm (center-to-center) spacing. The multiemitter device has a low dead volume to facilitate coupling to capillary liquid chromatography (LC) separations. The high aspect ratio of the emitters enables operation at flow rates as low as 20 nL/min/emitter, effectively extending the benefits of nanoelectrospray to higher flow rate analyses. To accommodate the larger ion current produced by the emitter array, a multicapillary inlet to the mass spectrometer was also constructed. The inlet, which matched the dimensions of the emitter array, preserved ion transmission efficiency. Standard reserpine solutions of varying concentration were electrosprayed at 1 microL/min using the multiemitter/multi-inlet combination, and the results were compared to those from a standard, single-emitter configuration. A 9-fold sensitivity enhancement was observed for the multiemitter relative to the single emitter. A bovine serum albumin tryptic digest was also analyzed, and a sensitivity increase ranging from 2.4- to 12.3-fold for the detected tryptic peptides resulted; the varying response was attributed to reduced ion suppression under the nanoESI conditions afforded by the emitter array. An equimolar mixture of leucine enkephalin and maltopentaose was studied to verify that ion suppression is indeed reduced for the multiplexed ESI (multi-ESI) array relative to a single emitter over a range of flow rates.     

Fabrication of internally tapered capillaries for capillary electrochromatography electrospray ionization mass spectrometry

In this study, we report a novel procedure for fabricating internally tapered capillary columns suitable for the coupling of capillary electrochromatography (CEC) to electrospray mass spectrometry (ESI-MS). The internal tapers were prepared by slowly heating the capillary end in a methane/O2 flame. Due to continuous self-shrinking of the inner channel of the capillary, the inside diameter of the opening was reduced to 7-10 microm. The procedure is easy to handle, with no requirement for expensive equipment as well as elimination of problematic grinding of the tip. Several advantages of these new internal tapers, as compared to using externally tapered columns, are described. First, the problems of poor durability and tip breakage associated with external tapering were successfully overcome with the internal taper. A comparison of the online CEC/ESI-MS between external versus internal tapers showed that the latter provides enhanced electrospray stability, resulting in significantly lower short-term noise and very short-term noise values. In turn, the more rugged design of internal tapers allows performing CEC/MS utilizing a harsh polar organic mobile phase, which was not previously successful using an external taper due to higher operating current and electrospray arcing. Next, data on the reproducibility of the internally tapered CEC/MS column using warfarin and beta-blockers as model analytes are presented. For example, when comparing the reproducibility for separation of warfarin under reversed-phase conditions, the internal taper demonstrated superior intraday % RSD (1.6-3.4) as compared to the external taper intraday % RSD (5-6). Last, the applicability of performing quantitative CEC/MS with internally tapered capillaries is demonstrated for simultaneous enantioseparation of beta-blockers. Impressive quantitative results include good linearity of calibration curves (e.g., R2 = 0.9940-0.9988) and limit of detection as low as 30 nM. The sensitive detection of a minor impurity of one enantiomer at the 0.1% level in a major chiral entity buttresses the suitability of compliance with FDA guidelines.     

Open-tubular capillary electrochromatography coupled with electrospray ionization mass spectrometry for peptide analysis

In this study, the open-tubular electrochromatographic (OT-CEC) migration behavior of various peptides has been characterized using etched and chemically (n-octadecyl- and cholesterol-) modified capillaries, interfaced to an electrospray ionization mass spectrometer through a sheath liquid configuration. The stationary phases were fabricated by etching the inner wall of the fused-silica capillary and then chemically modifying the new surface through a silanization/hydrosilation reaction. Unlike some other OT-CEC stationary-phase preparation methods, leaching of the immobilized stationary phase and subsequent contamination of the electrospray ion source was largely avoided with this novel surface modification technology. The influence of the immobilized organic phases and those of the buffer electrolytes (pH, the type and content of organic solvent) on the retention and separation of the selected peptides was investigated. Significant peptide retention was found even at very low pH with both types of stationary phases, under conditions whereby the electrophoretic migration dominated the separation process. Due to the effective coverage of the etched surface by a silanization/hydrosilation reaction, adverse adsorption of charged analytes onto the capillary wall was minimized. As a result, very efficient and highly reproducible peptide separations were achieved over a broad pH range. Moreover, peptide-specific multizoning effects were observed. The origin of this novel phenomenon was explored. Compared to capillary electrophoresis electrospray ionization mass spectrometry system, much higher detection sensitivity could be obtained, since a larger amount of sample could be injected and stacked at the head of the open-tubular capillary column without deteriorating the separation performance. On the basis of these observations, these procedures have been adapted to allow the analysis of tryptic peptides generated from proteins.     

Development of submillisecond time-resolved mass spectrometry using desorption electrospray ionization

Reaction kinetics studied by mass spectrometry (MS) has previously been limited to millisecond time resolution. This paper presents the development of a submillisecond time-resolved mass spectrometric method for fast reaction kinetic study, based on the capability of desorption electrospray ionization (DESI) for direct and fast ionization of a high-speed liquid jet stream. The principle underlying this methodology is that two reactant solutions undergo rapid mixing to produce a free liquid jet which is ionized by DESI at different positions corresponding to different reaction times. Due to the high velocity of the liquid jet, high time resolution can be achieved. In this study, the fast reduction reaction of 2, 6-dichlorophenolindophenol (DCIP) and L-ascorbic acid (L-AA) was chosen as an example to demonstrate this concept, and the reaction rate constant was successfully measured with an unprecedented time resolution of 300 μs. The good agreement of the measured value of (116 ± 3) s(-1) with that measured by the stopped-flow optical method (105 ± 2) s(-1) validates the feasibility of such a DESI-MS approach. Unlike classical spectroscopic techniques that require either chromophoric substrates or labeling, MS is a general detector with high chemical specificity. Therefore, this time-resolved DESI-MS method should find wide applications in fast (bio)chemical reaction investigations.     

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.     

Thin-layer chromatography and mass spectrometry coupled using desorption electrospray ionization

Desorption electrospray ionization (DESI) was demonstrated as a means to couple thin-layer chromatography (TLC) with mass spectrometry. The experimental setup and its optimization are described. Development lanes were scanned by moving the TLC plate under computer control while directing the stationary DESI emitter charged droplet plume at the TLC plate surface. Mass spectral data were recorded in either selected reaction monitoring mode or in full scan ion trap mode using a hybrid triple quadrupole linear ion trap mass spectrometer. Fundamentals and practical applications of the technique were demonstrated in positive ion mode using selected reaction monitoring detection of rhodamine dyes separated on hydrophobic reversed-phase C8 plates and reversed-phase C2 plates, in negative ion full scan mode using a selection of FD&C dyes separated on a wettable reversed-phase C18 plate, and in positive ion full scan mode using a mixture of aspirin, acetaminophen, and caffeine from an over-the-counter pain medication separated on a normal-phase silica gel plate.