Design and performance of a low-flow capillary electrophoresis-electrospray-mass spectrometry interface using an emitter with dual beveled edge

A low-flow electrophoresis-mass spectrometry (CE-MS) interface has been developed for interfacing capillary zone electrophoresis (CZE) with electrospray- ionization-mass spectrometry (ESI-MS). The interface consists of two parallel capillary columns (a separation column and a makeup column), and an emitter with a dual beveled edge. While maintaining a relatively low optimum flow rate, the dual-beveled-edge ESI emitter allows the use of a tip with larger orifice. Therefore, this interface is less prone to column blocking in comparison with a flat tip. Primarily attributed to low sample dilution and smaller initial droplet, the interface showed better sensitivity than a conventional sheath liquid interface. Furthermore, the interface was found to be more resistant to the presence of nonvolatile salts. By using 40 mM borate and 20 mM alpha-cyclodextrin (alpha-CD) as the running buffer, four major forms of gangliosides were detected by CE-MS.     

Microelectrospray interface with coaxial sheath flow for high-resolution capillary electrophoresis/mass spectrometry separation

We have fabricated a coaxial sheath liquid flow microelectrospray ionization (microESI) interface for capillary electrophoresis coupled with mass spectrometry (CE/MS). The ESI interface, which features a reduced probe diameter (130 microm i.d. x 174 microm o.d.) with a nebulizer-free format, can relatively easily electrospray a large amount of make-up sheath liquid (5-10 microL/min) over the long term (more than 80 runs) with a high degree of stability. The interface also provides higher separation qualities and improved detection sensitivities compared with a conventional ion spray (IS) interface.     

Influence of capillary dimensions on the performance of a coaxial capillary electrophoresis-electrospray mass spectrometry interface

The dimensions of the capillaries used to construct a typical coaxial capillary electrophoresis-mass spectrometry (CE-MS) interface, i.e. the inner diameter, the outer diameter and the wall thickness, have been shown to affect the performance of the CE-MS system. The influence of these parameters has been investigated in both MS and MS-MS modes. The initial results indicate that by reducing all the sheath capillaries' dimensions and the CE capillary outer diameter, better operation and increased sensitivity can be achieved. The capillary arrangement which gives optimum sensitivity and stable operation has been suggested.     

Modified liquid junction interface for nonaqueous capillary electrophoresis-mass spectrometry

Electrospray ionization (ESI) is the most widely used ionization method in on-line coupling of capillary electrophoresis-mass spectrometry (CE-MS). The conventional coaxial sheath flow electrospray interface is currently being replaced by the more sensitive nanoelectrospray technique. The usual limitation of nanoelectrospray CE-MS interface has been its short lifetime caused by deterioration of the metal coating on the CE capillary terminus. This article describes an easy way to construct a more durable and sensitive nanospray interface for nonaqueous CE-MS. In this approach a very thin glass spray capillary (ca. 30 microm outer diameter) is partly inserted inside the CE capillary, the junction being surrounded by the electrolyte medium, which is in contact with the platinum electrode. The interface was tested with five pharmaceuticals: methadone, pentazocine, levorphanol, dihydrocodeine, and morphine. Detection limits ranged from 12 to 540 fmol. Separation efficiency and reproducibility were also studied. The CE current was found to be stable and the migration times were highly reproducible. All the CE separations were carried out in a nonaqueous background electrolyte solution.     

A robust micro-electrospray ionization technique for high-throughput liquid chromatography/mass spectrometry proteomics using a sanded metal needle as an emitter

A 60 microm internal diameter (i.d.) stainless-steel needle was adapted to the orthogonal ESI probe ( microESI) of a commercial ion trap mass spectrometer, and used for capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) protein identification experiments. The modification allows for the use of nitrogen sheath gas which helps in the nebulization at LC flow rates exceeding 500 nL/min and eliminates problems caused by liquid junctions commonly used to initiate nanospray ionization (NSI). A comparison is made between the performance of a 75 microm i.d. column with a 15 microm pulled glass tip using a liquid junction, and that of a 150 microm i.d. column using the new microESI device. The combination of the 150 microm i.d. column and microESI gave sensitivity close to that of NSI (250 attomoles horse heart myoglobin digest on column), and proved to be more robust than the standard pulled glass tips of similar i.d. No evidence of metal needle catalyzed oxidation of methionine was observed during analysis of the tetrapeptide MRFA under a range of test conditions. Phosphorylated peptides in a beta-casein tryptic digest were also successfully identified using the microESI interface with a steel needle. In addition it was found that a mild sanding of the metal needle tip improved spray performance.     

Design, optimisation, and evaluation of a sheath flow interface for automated capillary electrophoresis-electrospray-mass spectrometry

A sheath-flow capillary electrophoresis-mass spectrometry (CE-MS) system utilizing a fully integrated large-bore stainless-steel emitter electrode tapered at the end for micro-ionspray operation has been developed and evaluated. A separation capillary with an outer diameter of up to 360 microm was inserted into the electrode thus forming a void volume of less than 15 nL between the capillary end and the electrospray ionisation (ESI) tip. The sheath liquid, usually methanol-water (80:20) with 0.1% formic acid for positive ion mode or methanol for negative ion mode, was delivered at 0.5-1.0 microL/min. Unlike previously reported CE-MS interfaces, the CE-MS probe was incorporated directly onto an Applied Biosystems/MDS SCIEX orthogonal-spray Turbo "V" ion source for ease of use and automatic operation. This integration enables fast and facile coupling and replacement of the separation capillary without interrupting the ion source configuration, and the sheath liquid supply. The reusable electrospray electrode was precisely fabricated and aligned with the length of the nebulizing gas tube for improved reproducibility. Automation was achieved through software control of both CE and tandem MS (MS/MS) for unattended batch sample analysis. The system was evaluated for attomole- to low femtomole-level profiling of model peptides and protein mixtures, bisphosphates, as well as antiviral nucleosidic drugs in cellular extracts.     

A sheath-flow nanospray interface for capillary electrophoresis/mass spectrometry

We have developed a novel sheath-flow interface for low-flow electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The interface is composed of two capillaries. One is a tapered fused-silica ESI emitter suitable for microliter and nanoliter flow rate electrospray and the other is a tail-end gold-coated CE separation column that is inserted into the emitter. A sheath liquid is supplied between the column and the emitter capillaries. The gold coating and the sheath liquid are used as the conducting media for ESI and the CE circuit. This novel design was initially evaluated by an infusion ESI-MS analysis of the most common antiretroviral dideoxynucleosides, followed by CE/MS coupling analysis of several antidepressant drugs. With infusion studies, the effects of the sheath liquid and the sample flow rates on detection sensitivity and signal stability were investigated. For an emitter with an internal diameter of 30 microm, the optimum flow rates for the sheath and the sample were 200 and 300 nL/min, respectively. The main improvement of this approach in comparison with conventional sheath liquid approaches using an ionspray interface is the gain in sensitivity. Sensitivities were three times better for dideoxynucleosides analyzed by infusion and 12 times higher for antidepressant drugs analyzed by CE/MS with this interface compared with ionspray. The emitter is durable, disposable, and simple to fabricate.     

Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing

A new fraction collection system for capillary zone electrophoresis (CZE) and capillary isolelectric focusing (CIEF) is described. Exact timing of the collector steps was based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. Determination of the zone velocity shortly before collection overcame the need for constant analyte velocity throughout the column. Consequently, sample stacking in CZE with large injection volumes as well as zone focusing in CIEF could be utilized with high collection accuracy. Capillaries of 200 microm inner diameter (ID) were employed in CZE and 100 microm ID in CIEF for the micropreparative mode. A sheath flow fraction collector was used to maintain permanent electric current during the collection. The bulk liquid flow due to siphoning, as well as the backflow arising from the sheath flow droplet pressure, were suppressed by closing the separation system at the inlet with a semipermeable membrane. In the CZE mode, the performance of the fraction collector is demonstrated by isolation of individual peaks from a fluorescently derivatized oligosaccharide ladder. In the CIEF mode, collection of several proteins from a mixture of standards is shown, followed by subsequent analysis of each protein fraction by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Coupling of a large-size capillary column with an electrospray mass spectrometer. A reliable and sensitive sheath flow capillary electrophoresis-mass spectrometry interface

The concept of interfacing a large-size column for capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS) for robust and automatic CE-MS operation is reported. Both standard ionspray interface and microionspray interface have been modified to operate in a sheath flow pattern to overcome the common stability problem in CE-MS coupling. To make the interface sensitive, a step-down stainless steel tube with smaller inner diameter and tapered tip was incorporated onto a larger tube embracing the CE column via cold soldering. The devices were evaluated for quantitative analysis of nucleotides at femtomole level and stable analytical performance in peptide profiling.     

Laser spray: electric field-assisted matrix-assisted laser desorption/ionization

A new liquid chromatography/mass spectrometry interface, the laser spray, has been developed. Explosive vaporization and mist formation occur when an aqueous solution effusing out from the tip of the stainless-steel capillary is irradiated from the opposite side of the capillary by a 10.6 microm infrared laser. Weak ion signals could be detected when the plume was sampled through the ion sampling orifice. When a high voltage (3-4 kV) was applied to the stainless-steel capillary, strong ion signals appeared. The ion abundances were found to be orders of magnitude greater than those obtained by conventional electrospray ionization in the case of aqueous solutions. The present method is regarded as an electric-field assisted form of matrix-assisted laser desorption/ionization in which the liquid chromatographic solvent (water, etc.) acts as a liquid matrix. Laser spray ionization is expected to become a versatile method for biological mass spectrometry because this method is compatible with the natural solvent, water.     

A new mass spectrometry electrospray tip obtained via precise mechanical micromachining

A new electrospray tip incorporating a beveled edge has been designed for use in plastic electrophoresis chip/electrospray mass spectrometry. Theoretical hydrodynamic analysis has been conducted to model the analytical sensitivity of the spray tip shape. A larger sample wall, that provides increased hydraulic pressure, is recommended in order to preserve the fluid stability at the tip outlet. A polymer with better hydrophobic characteristics than glass was used for the spray tip in order to restrict moisture accumulation at the spray tip outlet. Experimental results demonstrate that the analytical sensitivity of the proposed scheme is better than that obtained using the flat-head tip. Although a tapered capillary tip is commonly used in electrophoresis chip/electrospray mass spectrometry, the proposed tip offers a similar sensitivity while being more rugged and durable than the conventional tapered capillary tip. The cost of our design is also much lower than conventional spray tips.     

A novel nanoflow interface for atmospheric pressure ionization mass spectrometry

A novel spray-ionization technique for nanoflow liquid chromatography/mass spectrometry (nLC/MS) has been developed by modifying the sonic spray ionization (SSI) technique. A solution from a tapered fused-silica capillary is sprayed by a gas flow coaxial to the capillary, and ions produced are analyzed with an ion-trap mass spectrometer. The ion intensity is shown to have a steep threshold at a low gas velocity and to be much less dependent on the gas velocity than that of conventional SSI, in which the ion intensity is strongly dependent on the gas velocity and reaches its maximum at sonic velocity. Thus, we conclude that the concentration of charge in the solution at the tapered capillary tip with an inner diameter of 15 microm is almost at saturation so that charged droplets are produced from the solution by electrical force, rather than by sheer stress due to the gas flow. The ions are readily produced from these charged droplets. Preliminary results are compared with results obtained with a miniaturized electrospray unit.     

Analysis of low-molecular-mass organic acids using capillary zone electrophoresis-electrospray ionization mass spectrometry

A capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) method was developed to facilitate identification and determination of eleven low-molecular-mass (LMW) organic acids (i.e. oxalic, lactic, malonic, maleic, citric, tartaric, adipic, glutaric, gluconic, isosaccharinic and succinic acid) in different sample matrices. This CZE method was adapted to suit MS conditions. Sheath liquid, sheath flow and MS parameters were optimized to achieve high mass spectrometric sensitivity. The CZE-ESI-MS procedure showed good sensitivity (limit of detection of < 0.05-0.7 mg/l for all acids), linearity (r2 = 0.9925-0.9998) and reproducibility (2.09-5.34% RSD). The applicability of the CZE-ESI-MS was demonstrated on LMW organic acids in an ale sample. In addition the (here presented) method also provided quantification of fumaric, galacturonic and 2-ketoglutaric acid with high sensitivity.     

Interface for coupling nonaqueous wide-bore capillary electrophoresis with mass spectrometry

A liquid-junction-type interface where a thin spraying capillary is inserted inside the separation capillary was constructed for coupling nonaqueous wide-bore capillary electrophoresis (CE) to mass spectrometry (MS). The robust structure of the interface provided fairly easy capillary handling. The study was carried out with uncoated CE capillaries of 200 and 320 microm inner diameter (ID). 1-Propanol-acetonitrile (80:20 v/v) with acetate electrolyte provided a low conducting medium for CE and good spraying conditions for electrospray ionization (ESI) without sheath-flow and drying gas. Methamphetamine, alprenolol, and levorphanol served as model compounds. Approximate detection limits with the 200 microm ID capillary were 35-265 ng/mL.     

Performance of a sheathless porous tip sprayer for capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins

The performance of a prototype porous tip sprayer for sheathless capillary electrophoresis-mass spectrometry (CE-MS) of intact proteins was studied. Capillaries with a porous tip were inserted in a stainless steel needle filled with static conductive liquid and installed in a conventional electrospray ionization (ESI) source. Using a BGE of 100 mM acetic acid (pH 3.1) and a positively charged capillary coating, a highly reproducible and efficient separation of four model proteins (insulin, carbonic anhydrase II, ribonuclease A and lysozyme) was obtained. The protein mass spectra were of good quality allowing reliable mass determination of the proteins and some of their impurities. Sheath-liquid CE-MS using the same porous tip capillary and an isopropanol-water-acetic acid sheath liquid showed slightly lower to similar analyte responses. However, as noise levels increased with sheath-liquid CE-MS, detection limits were improved by a factor 6.5-20 with sheathless CE-MS. The analyte response in sheathless CE-MS could be enhanced using a nanoESI source and adding 5% isopropanol to the BGE, leading to improved detection limits by 50-fold to 140-fold as compared to sheath liquid interfacing using the same capillary - equivalent to sub-nM detection limits for three out of four proteins. Clearly, the sheathless porous tip sprayer provides high sensitivity CE-MS of intact proteins.     

Lactone cleavage reaction kinetics of rhodamine dye at liquid/liquid interfaces studied by micro-two-phase sheath flow/two-photon excitation fluorescence microscopy

Two-photon excitation fluorescence microscopy was combined with the two-phase microflow system in order to measure the fast interfacial reaction rate at liquid/liquid interfaces. The lactone cleavage kinetics of octadecylrhodamine B (C(18)RB) at the toluene/water and heptane/water interfaces was studied by this new method. The organic solution containing the nonfluorescent lactone of C(18)RB was made to flow as an inner flow with an aqueous outer sheath flow. The diameter of the inner flow was <20 microm. A focused fundamental beam of a Ti:sapphire pulse laser of 780 nm was irradiated to the interface, and emitted fluorescence from the fluorescent product was detected by a charge-coupled device (CCD) camera or a streakscope. The increase in the concentration of the fluorescent form of C(18)RB was measured along the interface of the inner flow of the toluene/water and heptane/water systems for 80 micros just after the contact of two phases. The analysis made by the time-dependent Langmuir adsorption model with the aid of the digital simulation method gave the cleavage reaction rate constants of the lactone form of C(18)RB at the liquid/liquid interfaces.     

A promising capillary electrophoresis–electrospray ionization–mass spectrometry method for carbohydrate analysis

A method for adapting widely used CE conditions for the separation of fluorescently labeled carbohydrates to permit online ESI‐MS detection is presented. Reverse polarity separations were performed in bare fused‐silica capillaries with an acidic BGE. Under these conditions, negatively charged 8‐aminopyrene 1,3,6‐trisulfonate‐labeled carbohydrates migrate forward against the EOF, which is towards the capillary inlet. Therefore, the CE‐MS interface must simultaneously back‐fill the capillary, in order to maintain the CE circuit, and provide a stable forward flow at the sprayer tip to support the electrospray process. This was achieved using a junction‐at‐the‐tip interface, which provides a flow of solution to the junction formed by the capillary terminus and the inner wall of the emitter needle tip. Because the flow rate required for this arrangement is much less than in conventional sheath flow interfaces, dilution of the analytes is minimized. Optimized separation conditions permit baseline resolution of glucose oligomers containing up to 15 glucose units, while longer oligomers, up to 33 glucose units, were observed as resolved peaks in the negative ion mode mass spectrum.     

CE-ESI-MS of biological anions in plastic capillaries at high pH

In this study, the potential of poly(methylmethacrylate) (PMMA, Plexiglas) and polyether ether ketone (PEEK) tubing for CE-ESI-MS separations of anions at high pH values was examined. A set of model compounds of biological interest was used to investigate the main operational parameters for CE-ESI-MS, such as the sheath-flow interface design, the polarity of the ionization voltage, the use of ammonia-based separation electrolytes, and the sheath liquid composition. Optimum separations and detection sensitivities in negative ESI mode were obtained using a running electrolyte of 75 mM of ammonia at pH 11 and a sheath liquid of 60:40 v/v or 75:25 v/v isopropanol/water with 0.5% v/v of ammonia. At these experimental conditions, PMMA and PEEK capillaries show good hydrolytic stabilities and lower EOF values than fused-silica columns. Better separation resolutions were obtained with PMMA capillary, but this plastic rapidly swelled and bled because of its limited chemical resistance to the sheath liquid. PMMA columns equipped with a fused-silica tip were used for a safer exposure to the sheath liquid, but the inner surface of the fused-silica tips had limited stability at pH 11. On the other hand, good separations and reproducibility on migration times and peak areas were obtained using PEEK capillaries without capillary column deterioration.     

Use of a fritless dual tapered column and a low flow interface for capillary electrochromatography-mass spectrometry

To avoid problems associated with the use of sintered frits to retain packing material, tapered columns were investigated for use with capillary electrochromatography-mass spectrometry (CEC-MS) analysis. Taking the advantage that negatively charged stationary phase particles have a net velocity directed towards the buffer reservoir (inlet) over a wide range in pH, a fritless CEC column with a single taper tip was prepared for CEC-MS analysis. During CEC-MS analysis, the tapered end was immersed in the buffer reservoir and the unmodified end was pointed toward the ionization source. For better sensitivity, this single tapered CEC column was coupled to ESI/MS using a low flow sheath liquid interface. With this setup, occasional blockage of the ESI sprayer by stationary phase particles was observed. In addition, significant dead volume was observed because the unmodified tip could not be inserted into the very end of the sprayer of the low flow sheath liquid interface. To circumvent these problems, a dual tapered CEC column was prepared. This fritless dual tapered column CEC-MS approach alleviated the problems of frit, sprayer blockage and extensive dead volume.     

Development of a capillary electrophoresis-mass spectrometry method for the determination of rivastigmine in human plasma--optimization of the limits of detection and quantitation

A capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) method was developed for the analysis of the acetylcholinesterase inhibitor rivastigmine. Several electrophoretic and ESI-MS parameters were investigated in order to improve sensitivity. These parameters were categorized in three areas: (i) background electrolyte (BGE) parameters, (ii) sheath liquid parameters, and (iii) spray chamber parameters. The optimized results were obtained by using 40-mM ammonium acetate at pH 9 as BGE, a sheath liquid of 1% acetic acid in water:MeOH (50:50 v/v) at a flow rate of 10?μL/min, and a drying gas flow rate that was set at 6 L/min and at a temperature of 200°C. These parameters provided limit of detection and limit of quantitation of 2.8?ng/mL and 8.4?ng/mL, respectively. The optimal CZE-ESI-MS conditions were applied to a plasma sample obtained from an Alzheimer's disease patient following rivastigmine patch administration, and the mean (±standard deviation) plasma concentration was estimated to be 14.6 (±1.7)?ng/mL. Several sample preparation procedures were examined, and solid-phase extraction using a C18 cartridge proved to be the most effective procedure, since higher sensitivity and recovery were obtained. In addition, precision was evaluated based on migration time and peak area in plasma, and the relative standard deviations were in the range of 0.10-0.16% and 0.62-9.0%, respectively.