Automation of nanoscale microcapillary liquid chromatography-tandem mass spectrometry with a vented column

To fully automate the sample introduction step for nanoscale microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, 75 microm i.d. x 14 cm capillary columns were interfaced with a commercial autosampler instrument using a novel procedure which allowed dilute peptide samples to be transferred from the AS loop injector to the nanoscale column at flow rates up to 5 microL min(-1). On-column enrichment and desalting was demonstrated for large sample volumes (>40 microL) by constructing a vent 2 cm after the entrance to the packed bed of 5-microm ODS-AQ modified silica. Salts and nonretained solutes were removed via the vent, which allowed for column washing independent of the continuation of the bed into the electrospray source. Separations of test peptide mixtures demonstrated 50-nL elution peak volumes with low- to subfemtomole detection levels. In addition, a highly complex peptide mixture (outer membrane preparation from Psuedemonas aeruginosa) was efficiently separated with more than 100 proteins identified from a single reversed-phase LC-MS/MS analysis. Finally, the vented column (V-column) was utilized for on-line separations in a multidimensional chromatography/tandem MS experiment where large numbers of strong cation exchange chromatography fractions from a trypsinized yeast lysate were desalted, concentrated, and analyzed in a completely automated fashion. The procedures for constructing and using a V-column require minimal changes in current methods and equipment for nano-LC-MS analyses using columns of 100-microm diameter and smaller.     

Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurements

The throughput of proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations as well as the subsequent mass spectrometric analysis; at present, analysis times can range anywhere from hours (high throughput) to days or longer (low throughput). We have explored the basis for proteomics analyses conducted on the order of minutes using high-speed capillary RPLC combined through on-line electrospray ionization interface with high-accuracy mass spectrometry (MS) measurements. Short 0.8-microm porous C18 particle-packed 50-microm-i.d. capillaries were used to speed the RPLC separations while still providing high-quality separations. Both time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) MS were applied for identifying peptides using the accurate mass and time (AMT) tag approach. Peptide RPLC relative retention (elution) times that were generated by solvent gradients that differed by at least 25-fold were found to provide relative elution times that agreed to within 5%, which provides the basis for using peptide AMT tags for higher throughput proteomics measurements. For fast MS acquisition speeds (e.g., 0.2 s for TOF and either approximately 0.3 or approximately 0.6 s for FTICR), peptide mass measurement accuracies of better than +/-15 ppm were obtained with the high-speed RPLC separations. The ability to identify peptides and the overall proteome coverage was determined by factors that include the separation peak capacity, the sensitivity of the MS (with fast scanning), and the accuracy of both the mass measurements and the relative RPLC peptide elution times. The experimental RPLC relative elution time accuracies of 5% (using high-speed capillary RPLC) and mass measurement accuracies of better than +/-15 ppm allowed for the confident identification of >2800 peptides and >760 proteins from >13,000 different putative peptides detected from a Shewanellaoneidensis tryptic digest. Initial results for both RPLC-ESI-TOF and RPLC-ESI-FTICR MS were similar, with approximately 2000 different peptides from approximately 600 different proteins identified within 2-3 min. For <120-s proteomic analysis, TOF MS analyses were more effective, while FTICR MS was more effective for the >150-s analysis due to the improved mass accuracies attained using longer spectrum acquisition times.     

Quantitative multiple reaction monitoring of peptide abundance introduced via a capillary zone electrophoresis-electrospray interface

We demonstrate the use of capillary zone electrophoresis with an electrokinetic sheath-flow electrospray interface coupled to a triple-quadrupole mass spectrometer for the accurate and precise quantification of Leu-enkephalin in a complex mixture using multiple-reaction monitoring (MRM). Assay time is <6 min, with no re-equilibration required between runs. A standard curve of Leu-enkephalin was performed in the presence of a background tryptic digest of bovine albumin. We demonstrate reasonably reproducible peak heights (21% relative standard deviation), retention times (better than 1% relative standard deviation), and robust electrospray quality. Our limit of detection (3σ) was 60 pM, which corresponds to the injection of 335 zmol of peptide. This is a 10-20-fold improvement in mass sensitivity than we have obtained by nano HPLC/MRM and substantially better than reported for LC/MS/MS. Further quantification was performed in the presence of stable-isotope-labeled versions of the peptides; under these conditions, linearity was observed across nearly 4 orders of magnitude. The concentration detection limit was 240 pM for the stable-isotope-labeled quantification.     

High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics

We describe high-efficiency (peak capacities of approximately 10(3)) nanoscale (using column inner diameters down to 15 microm) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74.5 microm were successfully packed with 3-microm C18-bonded porous (300-A pores) silica particles at a pressure of 18,000 psi. With a mobile-phase delivery pressure of 10,000 psi, these packed capillaries provided mobile-phase flow rates as low as approximately 20 nL/min at LC linear velocities of approximately 0.2 cm/s, which is near optimal for separation efficiency. To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 microm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 microm (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of approximately 10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-microm-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C18 particles with surface pores of 300-A versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC separations. Chromatographic elution time, MS response intensity, and mass measurement accuracy was examined between runs with a single column (with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples (various concentrations of cellular proteolytic peptides) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.     

High-efficiency on-line solid-phase extraction coupling to 15-150-microm-i.d. column liquid chromatography for proteomic analysis

The ability to manipulate and effectively utilize small proteomic samples is important for analyses using liquid chromatography (LC) in combination with mass spectrometry (MS) and becomes more challenging for very low flow rates due to extra column volume effects on separation quality. Here we report on the use of commercial switching valves (150-microm channels) for implementing the on-line coupling of capillary LC columns operated at 10,000 psi with relatively large solid-phase extraction (SPE) columns. With the use of optimized column connections, switching modes, and SPE column dimensions, high-efficiency on-line SPE-capillary and nanoscale LC separations were obtained demonstrating peak capacities of approximately 1000 for capillaries having inner diameters between 15 and 150 microm. The on-line coupled SPE columns increased the sample processing capacity by approximately 400-fold for sample solution volume and approximately 10-fold for sample mass. The proteomic applications of this on-line SPE-capillary LC system were evaluated for analysis of both soluble and membrane protein tryptic digests. Using an ion trap tandem MS it was typically feasible to identify 1100-1500 unique peptides in a 5-h analysis. Peptides extracted from the SPE column and then eluted from the LC column covered a hydrophilicity/hydrophobicity range that included an estimated approximately 98% of all tryptic peptides. The SPE-capillary LC implementation also facilitates automation and enables use of both disposable SPE columns and electrospray emitters, providing a robust basis for automated proteomic analyses.     

Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000-1500 and capabilities in proteomics and metabolomics

Proteomics analysis based-on reversed-phase liquid chromatography (RPLC) is widely practiced; however, variations providing cutting-edge RPLC performance have generally not been adopted even though their benefits are well established. Here, we describe an automated format 20 kpsi RPLC system for proteomics and metabolomics that includes on-line coupling of micro-solid phase extraction for sample loading and allows electrospray ionization emitters to be readily replaced. The system uses 50 microm i.d. x 40-200 cm fused-silica capillaries packed with 1.4-3-microm porous C18-bonded silica particles to obtain chromatographic peak capacities of 1000-1500 for complex peptide and metabolite mixtures. This separation quality provided high-confidence identifications of >12 000 different tryptic peptides from >2000 distinct Shewanella oneidensis proteins (approximately 40% of the proteins predicted for the S. oneidensis proteome) in a single 12-h ion trap tandem mass spectrometry (MS/MS) analysis. The protein identification reproducibility approached 90% between replicate experiments. The average protein MS/MS identification rate exceeded 10 proteins/min, and 1207 proteins were identified in 120 min through assignment of 5944 different peptides. The proteomic analysis dynamic range of the 20 kpsi RPLC-ion trap MS/MS was approximately 10(6) based on analyses of a human blood plasma sample, for which 835 distinct proteins were identified with high confidence in a single 12-h run. A single run of the 20 kpsi RPLC-accurate mass MS detected >5000 different compounds from a metabolomics sample.     

Primary structure determination of peptides and enzymatically digested proteins using capillary liquid chromatography/mass spectrometry and rapid linked-scan techniques

Primary protein sequences were determined for both peptides and enzymatically digested proteins by rapid linked-scan (B/E) liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) at the low-picomole level (10-50 pmol). During the course of a single LC/MS/MS analysis, we demonstrated that it is possible to generate interpretable collision-induced dissociation spectra of the eluting proteolytic peptides. Molecular weights of tryptic peptides were established by using 1/10 of the protein digest by operating in the capillary LC/frit-FABMS mode. Peptides exhibiting the strongest MH+ ions were then selected for subsequent LC/MS/MS analysis (typically 1/5 of the remaining protein digest). Elution times for each chromatographic peak were generally greater than 30 s. It was therefore possible to obtain a minimum of six B/E fast linked-scan spectra during the course of elution of each peptide component. Typically, B/E linked scans of the greatest ion abundance (obtained at the chromatographic peak maximum) were averaged to enhance the signal/noise ratio at these low-picomole levels. Unit resolution was observed for product ions below m/z 1000. Rapid linked scanning by LC/frit-FABMS/MS provided mass assignments for product ions within 0.2-0.3 amu of theoretical values. Side-chain fragment ions (wn and dn) were also observed, which allowed for the differentiation of isobaric amino acids (e.g., leucine and isoleucine). Examples of the application of this fast linked-scan technique to LC/MS/MS are presented for complex mixtures of unknown peptides and the tryptic digestion of phosphorylated beta-casein.     

A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics

One of the strategies of functional proteomics, research aiming to discover gene function at the protein level, is the comprehensive analysis of protein-protein interactions related to the functional linkage among proteins and analysis of functional cellular machinery to better understand the basis of cell functions. Here, we describe the direct nanoflow LC (DNLC) system, which is equipped with a fritless high-resolution electrospray interface column packed with 1-microm reversed-phase (RP) beads and a novel splitless nanoflow gradient elution system to operate the column. Using RP-DNLC at an extremely slow flow rate, <50 nL/min, combined with data-dependent collision-induced dissociation tandem MS (MS/MS) and computer-assisted retrieval of spectra, we identified approximately 100 protein components in a biological complex such as a premature mammalian ribosome pull-down from cultured cells when we used an epitope-tagged protein as bait. Because this analysis is most sensitive, requires approximately 0.2 microg of total protein, and is a fully automated 1-h process, we anticipated that it should be an excellent tool for analyzing a limited amount of functional multi-protein complexes in cells.     

Development of a nebulizer for a sheathless interfacing of NanoHPLC and ICPMS

A novel nebulizer (nDS-200) working at sample uptake rates of less than 500 nL min(-1) was developed for a sheathless interfacing of nanoHPLC (75-microm column i.d.) with ICPMS. It was based on a hollow fused-silica needle of which the tip (i.d. 10 microm, o.d. 20 microm) centered in a 254-microm-i.d. sapphire orifice. The nebulizer, equipped with a 3-cm(3) drain-free vaporization chamber, enabled a stable introduction into an ICP of aqueous mobile phases containing up to 95% acetonitrile at eluent flow rates between 50 and 450 nL min(-1). The low dead volume of the interface resulted in a peak width of 1.3 s (at half-height) and the entirely preserved chromatographic resolution. An example application of the coupling to the analysis of a tryptic digest of a SIP18 protein containing two to nine selenomethionine residues was described. The absolute detection limit was 25 fg (80Se), which allowed the detection of low-abundant selenopeptides at the femtomole level. In contrast to electrospray MS, the ICPMS detection in nanoHPLC is unaffected by the coeluting matrix and concomitant compounds and offers an elegant method for the detection and quantification of minor heteroelement-containing species prior to or in parallel with ESI MS analysis.     

MicroSPE-nanoLC-ESI-MS/MS using 10-microm-i.d. silica-based monolithic columns for proteomics

Silica-based monolithic capillary columns (25 cm x 10 microm i.d.) with integrated nanoESI emitters have been developed to provide high-quality and robust microSPE-nanoLC-ESI-MS analyses. The integrated nanoESI emitter adds no dead volume to the LC separation, allowing stable electrospray operation at flow rates of approximately 10 nL/min. In an initial application with a linear ion trap MS, we identified 5510 unique peptides that covered 1443 distinct Shewanella oneidensis proteins from a 300-ng tryptic digest sample in a single 4-h LC-MS/MS analysis. The use of an integrated monolithic ESI emitter provided enhanced resistance to clogging and provided good run-to-run reproducibility.     

Elastomeric microchip electrospray emitter for stable cone-jet mode operation in the nanoflow regime

Despite widespread interest in combining laboratory-on-a-chip technologies with mass spectrometry (MS)-based analyses, the coupling of microfluidics to electrospray ionization (ESI)-MS remains challenging. We report a robust, integrated poly(dimethylsiloxane) microchip interface for ESI-MS using simple and widely accessible microfabrication procedures. The interface uses an auxiliary channel to provide electrical contact for the stable cone-jet electrospray without sample loss or dilution. The electric field at the channel terminus is enhanced by two vertical cuts that cause the interface to taper to a line rather than to a point, and the formation of a small Taylor cone at the channel exit ensures subnanoliter postcolumn dead volumes. Cone-jet mode electrospray was demonstrated for up to 90% aqueous solutions and for extended durations. Comparable ESI-MS sensitivities were achieved using both microchip and conventional fused silica capillary emitters, but stable cone-jet mode electrosprays could be established over a far broader range of flow rates (from 50-1000 nL/min) and applied potentials using the microchip emitters. This attribute of the microchip emitter should simplify electrospray optimization and make the stable electrospray more resistant to external perturbations.     

Microchip electrospray: improvements in spray and signal stability during gradient elution by an inverted postcolumn makeup flow

Dynamic changes in mobile phase composition during high-performance liquid chromatography (HPLC) gradient elution coupled to mass spectrometry (MS) sensitively affect electrospray modes. We investigate the impact of the eluent composition on spray stability and MS response by infusion and injection experiments with a small tetrapeptide in water-acetonitrile mixtures. The employed HPLC/electrospray (ESI)-MS configuration uses a microchip equipped with an enrichment column, a separation column, and a makeup flow (MUF) channel. One nano pump is connected to the separation column, while a second one delivers solvent of exactly inverted composition to the MUF channel. Both solvent streams are united behind the separation column, before the ESI tip, such that the resulting electrosprayed solution always has identical composition during a gradient elution. Analyte peak parameters without and with MUF compensation are determined and discussed with respect to the electrospray mode and eluent composition. The postcolumn MUF significantly improves spray and signal stability over the entire solvent gradient, without compromising the performance of the HPLC separation column. It can also be conveniently implemented on microchip platforms.     

Screening and selective quantification of illicit drugs in wastewater by mixed-mode solid-phase extraction and quadrupole-time-of-flight liquid chromatography-mass spectrometry

For the first time, a mixed-mode solid-phase extraction with fractionation of basic analytes from neutral and acidic species during cartridge elution and liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) was combined for the quantitative determination of 24 illicit drugs and metabolites in urban sewage samples. The effects of several sample preparation and instrumental parameters in the sensitivity and selectivity of the quantitative method are thoroughly discussed. Under final working conditions, recoveries above 63% and 82% were attained for all species in raw and treated sewage, respectively; whereas, the limits of quantification of the method, defined for a signal-to-noise of 10 (S/N = 10), ranged from 2 to 50 ng L(-1). Sequential elution of mixed-mode cartridges allowed a significant reduction of matrix effects observed during electrospray ionization of basic drugs versus those measured for hydrophilic balance reversed-phase sorbents and the same mixed-mode polymer without fractionated elution. Analysis of raw wastewater samples confirmed the ubiquity of cocaine (COC), benzoylecgonine (BE), and 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in this matrix. The capability of the above methodology to identify new illicit drugs and/or metabolites in sewage samples is also discussed. With this aim, a two step strategy is proposed. First, high-resolution MS chromatograms, acquired throughout each chromatographic run, are automatically searched against an in-house built database, a reduced list of candidate drugs is generated, and the corresponding extracted ion chromatograms are obtained. In a further LC run, the tandem mass spectrometry (MS/MS) spectra of unknown peaks are acquired using different collision energies and compared with those existing in public libraries, or interpreted, to assign the unknown peak to one of the previously selected candidates.     

A micromachined chip-based electrospray source for mass spectrometry

A micromachining process is described for fabricating a mass spectrometry electrospray source on a silicon chip. The process utilizes polymer (parylene) layers to form a system of chambers, filters, channels, and hollow needle structures (electrospray emitters) that extend more than a millimeter beyond the edge of the silicon substrate. The use of photoresist as the sacrificial layer facilitates the creation of long channels. Access to the channel structures on the chip is through a port etched through the silicon substrate that also serves as a sample reservoir. A reusable chip holder consisting of two plastic plates and an elastomer gasket provides the means to mount the chip in front of the mass spectrometer inlet and make electrical and gas connections. The electrospray emitters have tapered tips with 5 microns x 10 microns rectangular openings. The shape of the tip can be varied depending on the shape of the mask used to protect the parylene structures during the final plasma etch. The parylene emitters are physically robust and require only a high electric field to achieve stable electrospray operation over a period of a few hours. Direct comparisons with conventional glass or fused silica emitters indicated very similar performance with respect to signal strength and stability, spectral quality, and endurance. The automated MS/MS analysis of a mixture of tryptic peptides was no more difficult and yielded nearly identical results as the analysis of the same sample using a conventional nanospray device. This work demonstrates that an efficient electrospray interface to mass spectrometry can be integrated with other on-chip structures and mass-produced using a batch process.     

A design for low-flow sheathless electrospray emitters

An extremely simple design has been developed for producing durable sheathless electrospray emitters that give highly stable electrospray for unlimited lifetimes. The emitters can be fashioned from any style fused-silica capillary and are ideally suited for generating "all-in-one" microcolumn-emitter systems thus eliminating unwanted void volumes. The emitters give stable electrospray at low (30 nL/min) as well as high (1 mL/min) flow rates without the aid of nebulizing gas. Fabrication of these emitters (aka the "fairy dust" technique) does not involve the use of a metallized coating but rather the adherance of 2-μm gold particles to the capillary tip resulting in a robust approach to the problem of making an electrical contact with the electrospray solvent.     

Microfluidic platform for liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures

A microfluidic chip that integrates all the fluidic components of a gradient liquid chromatography (LC) system is described. These chips were batch-fabricated on a silicon wafer using photolithographic processes and with Parylene as the main structural material. The fabricated chip includes three electrolysis-based electrochemical pumps, one for loading the sample and the other two for delivering the solvent gradient; platinum electrodes for delivering current to the pumps and establishing the electrospray potential; a low-volume static mixer; a column packed with silica-based reversed-phase support; integrated frits for bead capture; and an electrospray nozzle. The fabricated structures were able to withstand pressures in excess of 250 psi. The device was used to perform a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a mixture of peptides from the trypsin digestion of bovine serum albumen (BSA). Gradient elution through the 1.2-cm column was performed at a flow rate of 80 nL/min. Compared to the analysis of the same sample using a commercial nanoflow LC system, the chromatographic resolution was nearly as good, and the total cycle time was significantly reduced because of the minimal volume between the pumps and the column. Results demonstrate the potential of mass-produced, low-cost microfluidic systems capable of performing LC separations for proteomics applications.     

Direct MALDI-MS/MS of phosphopeptides affinity-bound to immobilized metal ion affinity chromatography beads

Immobilized metal ion affinity chromatography (IMAC) is a useful method to selectively isolate and enrich phosphopeptides from a peptide mixture. Mass spectrometry is a very suitable method for exact molecular weight determination of IMAC-isolated phosphopeptides, due to its inherent high sensitivity. Even exact molecular weight determination, however, is not sufficient for identification of the phosphorylation site if more than one potential phosphorylation site is present on a peptide. The previous method of choice for sequencing the affinity-bound peptides was electrospray tandem mass spectrometry (ESI-MS/MS). This method required elution and salt removal prior to MS analysis of the peptides, which can lead to sample loss. Using a matrix-assisted laser desorption/ionization (MALDI) source coupled to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer with true MS/MS capabilities, direct sequencing of IMAC-enriched peptides has been performed on IMAC beads applied directly to the MALDI target. The utility of this new method has been demonstrated on a protein with unknown phosphorylation sites, where direct MALDI-MS/MS of the tryptic peptides bound to the IMAC beads resulted in the identification of two novel phosphopeptides. Using this technique, the phosphorylation site determination is unambiguous, even with a peptide containing four potentially phosphorylated residues. Direct analysis of phosphorylated peptides on IMAC beads does not adversely affect the high-mass accuracy of an orthogonal injection QqTOF mass spectrometer, making it a suitable technique for phosphoproteomics.     

Screening for organic phosphorus compounds in aquatic sediments by liquid chromatography coupled to ICP-AES and ESI-MS/MS

The structures of organic phosphorous (P) compounds in aquatic sediments are to a large extent unknown although these compounds are considered to play an important role in regulating lake trophic status. To enhance identification of these compounds, a liquid chromatography (LC) method for their separation was developed. The stationary phase was porous graphitic carbon (PGC), and the mobile phases used in the gradient elution were compatible with both inductive coupled plasma atomic emission spectroscopy (ICP-AES) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). With LC-ICP-AES, eight different P containing peaks could be observed in the P chromatogram indicating that at least eight different P compounds were separated. With the setup of an information dependent acquisition (IDA) with ESI-MS/MS, the mass over charge ( m/ z) of compounds containing a phosphate group (H 2PO 3 (-), m/ z 97) could be measured and further fragmentation experiments gave additional information on the structure of almost 40 separated P compounds, several were verified to be nucleotides. ICP-AES was very suitable in the development of the LC method and allowed screening and quantification of P compounds. The presented LC-ESI-MS/MS technique was able to identify several sediment organic P compounds.     

Porous polymer monolith assisted electrospray

Coupling low-flow analytical separation instrumentation such as capillary electrophoresis, capillary electrochromatography, nano-HPLC, and microfluidic-based devices with electrospray ionization mass spectrometry has yielded powerful analytical tools. However, conventional coupling methodologies such as nanospray suffer from limitations including poor conductive coating robustness, constant clogging, complicated fabrication processes, and incompatibility with large flow rate regimes. This study demonstrates that robust nanospray emitters can be fabricated through the formation and utilization of a porous polymer monolith (PPM) at the end of a fused-silica capillary. Stable electrosprays can be produced from capillaries (75-100-microm i.d.) at a variety of flow rates (50-1000 nL/min) without the need to taper the capillaries by etching or pulling. The PPM is photopatterned to be present only near the capillary exit aperture using conditions that generate pore sizes similar to those seen with nanospray tips. The porous nature of the PPM aids in developing a stable electrospray generating a single clearly visible Taylor cone at relatively high flow rates while at low flow rates (<100 nL/min) a mist, presumably from multiple small Taylor cones, develops. The hydrophobic nature of the PPM should limit problems with band broadening associated with droplet spreading at the capillary exit, while the multiple flow paths inherent in the PPM minimize clogging problems associated with conventional nanospray emitters. Total ion current traces for a constant infusion of standard PPG and cytochrome c solutions are very stable with deviations ranging from only 3 to 8%. The PPM-assisted electrospray produces mass spectra with excellent signal-to-noise ratios from only a few femtomoles of material.     

Electrospray Ion Chromatography-Tandem Mass Spectrometry of Bromate at Sub-ppb Levels in Water

An electrospray ion chromatography-tandem mass spectrometry (IC-MS/MS) method has been developed for the analysis of bromate ions in water. This IC-MS/MS method improves the limit of detection of bromate ions by a factor of 10. The method consists of solid phase extraction with an ion exchange column and elution of the analyte with water/methanol ammonium sulfate eluent on-line with a negative ion electrospray mass spectrometry detection. SPE requires sample pretreatment to remove any major ions that displace bromate, consisting of eliminating SO(4)(2)(-), Cl(-), and HCO(3)(-) ions respectively with barium-form, silver-form, and acid (H(+)-form) exchange resins. The methanolic sulfate eluent permits IC-MS coupling via an electrospray interface. BrO(3)(-) was selected in the first quadrupole (Q1) at two m/z values, 127 and 129, according to the isotope contributions of (79)Br and (81)Br. After fragmentation in the collision cell (second quadrupole, Q2), the third quadrupole (Q3) analyzes the product ions as (M - O)(-), (M - 2O)(-), and (M - 3O)(-). Among the six recordable transitions, four were selected, the other two yielding high background. A lowered resolution raised sensitivity by a factor of up to 3. The limit of quantitation of this method was 0.1 μg/L.