Identification of fulvic acids and sulfated and nitrated analogues in atmospheric aerosol by electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

The water-soluble organic fractions of aerosol samples collected in Riverside, CA, in summer 2005 were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Elemental compositions of about 1000 molecular species were determined in the range m/z 220-420, and four series of organic compounds were identified, fulvic acids, and S-containing, N-containing, and S- and N-containing molecules. Low-resolution product ion spectra proved the presence of organosulfates, organonitrates, and mixed organosulfates and -nitrates that appear to be structurally closely related to each other and to the fulvic acids. This is the first unambiguous detection of fulvic acid molecules and sulfated components in atmospheric aerosol and the first detection even of nitrated analogues. These species provide new clues to the nature of particulate organic matter in atmospheric aerosol.     

Simple coupling of gas chromatography to electrospray ionization mass spectrometry

A simple method for direct coupling of gas chromatography (GC) with electrospray ionization mass spectrometry (ESI/MS) has been developed. The outlet of the GC capillary column was placed between the ESI needle and the atmospheric pressure ionization (API) source of a mass spectrometer. The ionization occurs via dissolution of neutral compounds into the charged ESI droplet followed by ion evaporation or via a gas-phase proton transfer reaction between a protonated solvent molecule and an analyte. The mass spectra of organic volatile compounds showed abundant protonated molecules with little fragmentation, being very similar to those produced by normal liquid ESI. The quantitative performance of the system was evaluated by determining the limit of detection (LOD), linearity ( r (2)), and repeatability (RSD). The GC-ESI/MS method was shown to be stable, providing high sensitivity and good quantitative performance.     

Selective ionization of dissolved organic nitrogen by positive ion atmospheric pressure photoionization coupled with Fourier transform ion cyclotron resonance mass spectrometry

Dissolved organic nitrogen (DON) comprises a heterogeneous family of organic compounds that includes both well-known biomolecules such as urea or amino acids and more complex, less characterized compounds such as humic and fulvic acids. Typically, DON represents only a small fraction of the total dissolved organic carbon pool and therefore presents inherent problems for chemical analysis and characterization. Here, we demonstrate that DON may be selectively ionized by atmospheric pressure photionization (APPI) and characterized at the molecular level by Fourier transform ion cyclotron resonance mass spectrometry. Unlike electrospray ionization (ESI), APPI ionizes polar and nonpolar compounds, and ionization efficiency is not determined by polarity. APPI is tolerant to salts, due to the thermal treatment inherent to nebulization, and thus avoids salt-adduct formation that can complicate ESI mass spectra. Here, for dissolved organic matter from various aquatic environments, we selectively ionize DON species that are not efficiently ionized by other ionization techniques and demonstrate significant signal-to-noise increase for nitrogen species by use of APPI relative to ESI.     

Ambient mass spectrometry with a handheld mass spectrometer at high pressure

The first coupling of atmospheric pressure ionization methods, electrospray ionization (ESI) and desorption electrospray ionization (DESI), to a miniature hand-held mass spectrometer is reported. The instrument employs a rectilinear ion trap (RIT) mass analyzer and is battery-operated, hand-portable, and rugged (total system: 10 kg, 0.014 m(3), 75 W power consumption). The mass spectrometer was fitted with an atmospheric inlet, consisting of a 10 cm x 127 microm inner diameter stainless steel capillary tube which was used to introduce gas into the vacuum chamber at 13 mL/min. The operating pressure was 15 mTorr. Ions, generated by the atmospheric pressure ion source, were directed by the inlet along the axis of the ion trap, entering through an aperture in the dc-biased end plate, which was also operated as an ion gate. ESI and DESI sources were used to generate ions; ESI-MS analysis of an aqueous mixture of drugs yielded detection limits in the low parts-per-billion range. Signal response was linear over more than 3 orders of magnitude. Tandem mass spectrometry experiments were used to identify components of this mixture. ESI was also applied to the analysis of peptides and in this case multiply charged species were observed for compounds of molecular weight up to 1200 Da. Cocaine samples deposited or already present on different surfaces, including currency, were rapidly analyzed in situ by DESI. A geometry-independent version of the DESI ion source was also coupled to the miniature mass spectrometer. These results demonstrate that atmospheric pressure ionization can be implemented on simple portable mass spectrometry systems.     

The use of multidimensional liquid-phase separations and mass spectrometry for the detailed characterization of posttranslational modifications in glycoproteins

The goal of characterization of the proteome, while challenging in itself, is further complicated by the microheterogeneity introduced by posttranslational modifications such as glycosylation. A combination of liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry (MS) offers the advantages of unique selectivity and high efficiency of the separation methods combined with the mass specificity and sensitivity of MS. In the current work, the combination of liquid-phase separations and mass spectrometry is demonstrated through the on-line coupling of electrospray ionization mass spectrometry (ESI-MS) and off-line coupling with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). LC/ESI-MS yields real-time results while maintaining the separation obtained from the LC analysis. CE/MALDI TOF-MS offers high-mass detection and extremely low detection limits. The unique separation selectivity of CE relative to reversed-phase HPLC separations of the members of a glycopeptide family was used to develop an integrated multidimensional analysis achieved by the off-line coupling of LC, CE, and MALDI TOF-MS. To demonstrate the applicability of these techniques to the characterization of the heterogeneity of posttranslational modifications present in glycoproteins, we will report on the study of the glycoforms present in a N-linked site in a single-chain plasminogen activator (DSPAα1).     

Zeptomole-sensitivity electrospray ionization--Fourier transform ion cyclotron resonance mass spectrometry of proteins

Methods are being developed for ultrasensitive protein characterization based upon electrospray ionization (ESI) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The sensitivity of a FTICR mass spectrometer equipped with an ESI source depends on the overall ion transmission, which combines the probability of ionization, transmission efficiency, and ion trapping in the FTICR cell. Our developments implemented in a 3.5 tesla FTICR mass spectrometer include introduction and optimization of a newly designed electrodynamic ion funnel in the ESI interface, improving the ion beam characteristics in a quadrupole-electrostatic ion guide interface, and modification of the electrostatic ion guide. These developments provide a detection limit of approximately 30 zmol (approximately 18,000 molecules) for proteins with molecular weights ranging from 8 to 20 kDa.     

Interpretation of mass spectra from organic compounds in aerosol time-of-flight mass spectrometry

Organic compounds containing a variety of functional groups have been analyzed using aerosol time-of-flight mass spectrometry. Both positive and negative laser desorption/ionization mass spectra have been acquired for compounds of relevance to ambient air particulate matter, including polycyclic aromatic hydrocarbons, heterocyclic analogues, aromatic oxygenated compounds such as phenols and acids, aliphatic dicarboxylic acids, and reduced nitrogen species such as amines. In many cases, positive ion mass spectra are similar to those found in libraries for 70-eV electron impact mass spectrometry. However, formation of even-electron molecular ions due to adduct formation also plays a major role in ion formation. Negative ion mass spectra suggest that organic compounds largely disintegrate into carbon cluster fragments (C(n)- and C(n)H-). However, information about the heteroatoms present in organic molecules, especially nitrogen and oxygen, is carried dominantly by negative ion spectra, emphasizing the importance of simultaneous analysis of positive and negative ions in atmospheric samples.     

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.     

Design and performance of a universal sheathless capillary electrophoresis to mass spectrometry interface using a split-flow technique

A split-flow capillary electrophoresis electrospray ionization mass spectrometry (CE/ESI-MS) interface is introduced, in which the electrical connection to the CE capillary outlet is achieved by diverting part of the CE buffer out of the capillary through an opening near the capillary outlet. The CE buffer exiting the opening contacts a sheath metal tube which acts as the CE outlet/ESI shared electrode. In cases in which the ESI source uses a metal needle, the voltage contact to the CE buffer is achieved by simply inserting the outlet of the CE capillary, which contains an opening, into the existing ESI needle (thereby greatly simplifying the CE to MS interfacing). As a result of the concentration-sensitive nature of ESI, splitting a small percentage of the CE flow has minimal effect on the sensitivity of detection. In addition, because the liquid is flowing through the opening and out of the capillary, there is no dead volume associated with this interface. Moreover, bubble formation due to redox reactions of water at the electrode does not effect CE/ESI-MS performance, because the actual metal/liquid contact occurs outside of the CE capillary. The sensitivity associated with a sheathless CE/MS interface, the ease of fabrication, universality, and lack of any dead volume make this design a superior CE/ESI-MS interface. The performance of this interface is demonstrated by analyses of a peptide standard and a protein digest using a variety of capillary dimensions.     

Secondary ionization of chemical warfare agent simulants: atmospheric pressure ion mobility time-of-flight mass spectrometry

For the first time, the use of a traditional ionization source for ion mobility spectrometry (radioactive nickel ((63)Ni) beta emission ionization) and three alternative ionization sources (electrospray ionization (ESI), secondary electrospray ionization (SESI), and electrical discharge (corona) ionization (CI)) were employed with an atmospheric pressure ion mobility orthogonal reflector time-of-flight mass spectrometer (IM(tof)MS) to detect chemical warfare agent (CWA) simulants from both aqueous- and gas-phase samples. For liquid-phase samples, ESI was used as the sample introduction and ionization method. For the secondary ionization (SESI, CI, and traditional (63)Ni ionization) of vapor-phase samples, two modes of sample volatilization (heated capillary and thermal desorption chamber) were investigated. Simulant reference materials, which closely mimic the characteristic chemical structures of CWA as defined and described by Schedule 1, 2, or 3 of the Chemical Warfare Convention treaty verification, were used in this study. A mixture of four G/V-type nerve simulants (dimethyl methylphosphonate, pinacolyl methylphosphonate, diethyl phosphoramidate, and 2-(butylamino)ethanethiol) and one S-type vesicant simulant (2-chloroethyl ethyl sulfide) were found in each case (sample ionization and introduction methods) to be clearly resolved using the IM(tof)MS method. In many cases, reduced mobility constants (K(o)) were determined for the first time. Ion mobility drift times, flight times, relative signal intensities, and fragmentation product signatures for each of the CWA simulants are reported for each of the methods investigated.     

Capillary electrophoresis to mass spectrometry interface using a porous junction

A new capillary electrophoresis interface to electrospray ionization mass spectrometry (CE/ESI-MS) is introduced in which the electrical connection to the CE capillary outlet/ESI electrode is achieved by transfer of small ions related to the background electrolyte (BGE) through a porous section near the CE capillary outlet. In this design, only a small section of the capillary wall is made porous. The porous section is created by first thinning a small section of the capillary wall by drilling a well into it and then etching the remaining thin wall porous. This design has two advantages over previous designs (in which the whole circumference of the capillary was made porous): first, the capillary interface is more robust because only a small section of it is made porous, and therefore, no liquid junction is needed to secure the porous section. The electrical connection is achieved simply by inserting the capillary outlet containing the porous junction into the existing ESI needle and filling the needle with the BGE. Second, the time required to make the fused silica porous is reduced from approximately 1 h to a few minutes. In addition, there is no dead volume associated with the porous design, and because the actual metal/liquid contact occurs outside of the CE capillary, bubble formation due to redox reactions of water at the electrode does not affect CE/ESI-MS performance. The performance of this interface is demonstrated by the analyses of peptide and protein mixtures.     

Molecular resolution and fragmentation of fulvic acid by electrospray ionization/multistage tandem mass spectrometry

Molecular weight distributions of fulvic acid from the Suwannee River, Georgia, were investigated by electrospray ionization/quadrupole mass spectrometry (ESI/ QMS), and fragmentation pathways of specific fulvic acid masses were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry (ESI/MST/ MS). ESI/QMS studies of the free acid form of low molecular weight poly(carboxylic acid) standards in 75% methanol/25% water mobile phase found that negative ion detection gave the optimum generation of parent ions that can be used for molecular weight determinations. However, experiments with poly(acrylic acid) mixtures and specific high molecular weight standards found multiply charged negative ions that gave a low bias to molecular mass distributions. The number of negative charges on a molecule is dependent on the distance between charges. ESI/MST/MS of model compounds found characteristic water loss from alcohol dehydration and anhydride formation, as well as CO2 loss from decarboxylation, and CO loss from ester structures. Application of these fragmentation pathways to specific masses of fulvic acid isolated and fragmented by ESI/MST/MS is indicative of specific structures that can serve as a basis for future structural confirmation after these hypothesized structures are synthesized.     

Gas chromatography connected to multiple channel electrospray ionization mass spectrometry for the detection of volatile organic compounds

This work successfully connected gas chromatography (GC) to seven-channel electrospray ionization (ESI) mass spectrometry to separate and detect a mixture of volatile organic compounds. Gaseous analyte was eluted separately from a GC column and directed into the central channel of the ESI source. The analyte was protonated by ion-molecule reactions between the analyte and the ions which were generated by electrospraying the acidic solution through the outside six channels surrounding the central channel. Real-time analysis of the organic reaction involving volatile and thermally unstable compounds (dimethylhydrazine ? azomethane + H(2)) was also achieved by continuously purging the air in the reaction vessel to the seven-channel ESI source.     

Multiple ionization mass spectrometry strategy used to reveal the complexity of metabolomics

A multiple ionization mass spectrometry strategy is presented based on the analysis of human serum extracts. Chromatographic separation was interfaced inline with the atmospheric pressure ionization techniques electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive (+) and negative (-) ionization modes. Furthermore, surface-based matrix-assisted laser desorption/ionization (MALDI) and desorption ionization on silicon (DIOS) mass spectrometry were also integrated with the separation through fraction collection and offline mass spectrometry. Processing of raw data using the XCMS software resulted in time-aligned ion features, which are defined as a unique m/z at a unique retention time. The ion feature lists obtained through LC-MS with ESI and APCI interfaces in both +/- ionization modes were compared, and unique ion tables were generated. Nonredundant, unique ion features, were defined as mass numbers for which no mass numbers corresponding to [M + H](+), [M - H](-), or [M + Na](+) were observed in the other ionization methods at the same retention time. Analysis of the extracted serum using ESI for both (+) and (-) ions resulted in >90% additional unique ions being detected in the (-) ESI mode. Complementing the ESI analysis with APCI resulted in an additional approximately 20% increase in unique ions. Finally, ESI/APCI ionization was combined with fraction collection and offline-MALDI and DIOS mass spectrometry. The parts of the total ion current chromatograms in the LC-MS acquired data corresponding to collected fractions were summed, and m/z lists were compiled and compared to the m/z lists obtained from the DIOS/MALDI spectra. It was observed that, for each fraction, DIOS accounted for approximately 50% of the unique ions detected. These results suggest that true global metabolomics will require multiple ionization technologies to address the inherent metabolite diversity and therefore the complexity in and of metabolomics studies.     

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.     

Flow-through microvial facilitating interface of capillary isoelectric focusing and electrospray ionization mass spectrometry

A capillary electrophoresis mass spectrometry (CE-MS) interface utilizing a flow-through microvial is used to ensure the electric continuity and supply the catholyte and mobilizer solutions during the capillary isoelectric focusing (cIEF) and mobilization process. The flow-through microvial provides a stable chemical environment and helps to improve the ionization efficiency without significantly diluting the analyte. The CE-MS interface facilitates the transfer of the mobilized cIEF effluent to the site of electrospray ionization, and the gaseous ions can be detected directly by a mass spectrometer. It also allows for complete focusing and mobilization processes to be performed automatically in programmed sequences with commercial CE systems. Two different strategies, using either a part of the capillary or the flow-through microvial of the CE-MS interface as the catholyte reservoir for bare fused silica capillaries or neutral coated capillaries, respectively, were developed for automated cIEF-electrospray ionization (ESI)-MS. Reasonable separation efficiency was achieved using proper concentration of carrier ampholytes and suitable strategies of electroosmotic/electrophoretic mobilization.     

Subambient pressure ionization with nanoelectrospray source and interface for improved sensitivity in mass spectrometry

A nanoelectrospray ionization mass spectrometry (ESI-MS) source and interface has been designed that enables efficient ion production and transmission in a 30 Torr pressure environment using solvents compatible with typical reversed-phase liquid chromatography (RPLC) separations. In this design, the electrospray emitter is located inside the mass spectrometer in the same region as an electrodynamic ion funnel. This avoids the use of a conductance limiting ion inlet, as required by a conventional atmospheric pressure ESI source, and allows more efficient ion transmission to the mass analyzer. The new subambient pressure ionization with nanoelectrospray (SPIN) source improves instrument sensitivity and enables new electrospray interface designs, including the use of multi-emitter approaches. Performance of the SPIN source was evaluated by electrospraying standard solutions at 300 nL/min and comparing results with those obtained from a standard atmospheric pressure ESI source that used a heated capillary inlet. This initial study demonstrated an approximately 5-fold improvement in sensitivity when the SPIN source was used compared to a standard atmospheric pressure ESI source. The importance of desolvation was also investigated by electrospraying at different flow rates, which showed that the ion funnel provided an effective desolvation region to aid the creation of gas-phase analyte ions.     

Secondary electrospray ionization ion mobility spectrometry/mass spectrometry of illicit drugs

A secondary electrospray ionization (SESI) method was developed as a nonradioactive ionization source for ion mobility spectrometry (IMS). This SESI method relied on the gas-phase interaction between charged particles created by electrospray ionization (ESI) and neutral gaseous sample molecules. Mass spectrometry (MS) was used as the detection method after ion mobility separation for ion identification. Preliminary investigations focussed on understanding the ionization process of SESI. The performance of ESI-IMS and SESI-IMS for illicit drug detection was evaluated by determining the analytical figures of merit. In general, SESI had a higher ionization efficiency for small volatile molecules compared with the electrospray method. The potential of developing a universal interface for both GC- and LC-MS with an addition stage of mobility separation was demonstrated.     

Design and characterization of a multisource hand-held tandem mass spectrometer

A wireless-controlled miniature rectilinear ion trap mass spectrometer system, total weight with batteries 5.0 kg, consuming less than 35 W of power, and having dimensions of 22 cm in length by 12 cm in width by 18 cm in height, is characterized. The design and construction of the mass spectrometer including mass analyzer, vacuum system, electronics system, and data acquisition and processing systems, is detailed. The mass spectrometer is compatible with various types of ionization sources including a glow discharge electron impact ionization source used in the internal ionization mode, and various atmospheric pressure ionization sources, including electrospray ionization, atmospheric pressure chemical ionization, and desorption electrospray ionization, which are employed for external, atmospheric pressure ionization. These external sources are coupled to the miniature mass spectrometer via a capillary interface that is operated in a discontinuous fashion (discontinuous atmospheric pressure interface) to maximize ion transport. The performance of the mass spectrometer for large and small molecules is characterized. Limits of detection in the parts-per-billion range were obtained for selected compounds examined using both the internal ionization and external ionization modes. Tandem mass spectrometry and fast in situ analysis capabilities are also demonstrated using a variety of compounds and ionization sources. Protein molecules are analyzed as the multiply protonated molecules with mass/charge ratios up to 1500 Da/charge.     

Simultaneous analysis of amino acids and carboxylic acids by capillary electrophoresis-mass spectrometry using an acidic electrolyte and uncoated fused-silica capillary

A simple, low-cost capillary electrophoresis-mass spectrometry (CE-MS) method is demonstrated for the simultaneous analysis of amino acids and small carboxylic acids (glycerate, lactate, fumarate, succinate, malate, tartrate, citrate, iso-citrate, cis-aconitate, and shikimate). All CE-MS experiments were performed using a single uncoated fused-silica capillary and with a single separation electrolyte, formic acid. For CE polarity, the CE inlet was set as the anode, and the MS side was set as the cathode. By using high-speed sheath gas flow, the apparent mobilities of all compounds were sped up; thus, the migration times of the carboxylic acids were reduced. In positive ion mode ESI-MS detection, small carboxylic acids were detected faintly as m/z = [M + 18](+) or [M + 23](+), after protonated molecule detection (m/z = [M + 1](+)) of the amino acids. In negative ion mode, all of these small carboxylic acids were detected clearly as deprotonated molecules (m/z = [M - 1](-)), after detection of the amino acids. By changing the polarity of the MS during CE separation, both amino acids and small carboxylic acids were detectable in a single electrophoresis analysis run. With this method, the diurnal metabolic changes of pineapple leaves were observed as reflecting Crassulacean acid metabolism.