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A novel electrochemical probe has been designed, built, and used to characterize the distribution in solution potential within the metal capillary and Taylor cone of the electrospray (ES) device. The measurement system consists of three electrodes--a counter electrode held at highly negative potential that serves as the cathode, and two anodes consisting of a disk-shaped, mobile, internal (working) electrode, and the internal surface of the surrounding ES capillary (auxiliary electrode, held at ground potential). One-dimensional differential electrospray emitter potential (DEEP) maps detailing solution potential gradients within the electrospray emitter and in the region of the Taylor cone are constructed by measuring the potential at the working electrode vs the ES capillary, as a function of working electrode position along the emitter axis. Results show that the measured potential difference increases as the internal probe travels toward the ES capillary exit, with values rising sharply as the base of the Taylor cone is penetrated. Higher conductivity solutions exhibit potentials of higher magnitude at longer distances away from the counter electrode, but these same solutions show lower potentials near the ES capillary exit. Removal of easily oxidizable species from the solution causes the measured potential difference to have nonzero values at distances further within the capillary, and the values measured at all points are raised. Results are consistent with the characterization of the electrospray system as a controlled-current electrolytic flow cell. Elucidation of the electrochemical details of the electrospray process can lead to mass spectrometric signal enhancement of certain species present in the spraying liquid and also allow the detection of molecules that are usually not observable due to their low ionization efficiencies.

The influence of the diameter of the spray tip employed for nano-electrospray mass spectrometry (nano-ES-MS) upon mass spectral charge state distributions was investigated using angiotensin I ([M.sub.r] = 1296), insulin ([M.sub.r] = 5774), and ubiquitin ([M.sub.r] = 8560) as test analytes. Under a variety of experimental conditions, the charge state distributions of the test peptides and protein consistently shifted toward higher values as the tip orifice diameter decreased. This finding indicates that the use of narrow diameter capillaries can promote the formation of higher charge state ions that are more reactive precursors in tandem mass spectrometry experiments. A detailed comparison of charge state distributions obtained for nanospray capillaries of varying diameters was undertaken while systematically varying experimental parameters such as sample flow rate, analyte concentration, solvent composition, and electrospray current. The general tendency to obtain higher charge states from narrow diameter capillaries was conserved throughout, but tips with smaller orifices were more sensitive to sample flow rate (the average charge state was lowered significantly as flow was raised), whereas tips with bigger orifices were more sensitive to analyte concentration and pH of the solution (as each was lowered, the average charge state increased).

The formation and decomposition (postsource decay, PSD) of anionic adducts in negative ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry have been studied. Chloride, a small inorganic anion, has been found to form stable anionic adducts with a variety of neutral oligosaccharides that can survive the MALDI process to give readily identifiable signals (with characteristic isotope patterns) allowing subpicomole detection in the best cases. The matrixes that can aid the formation of chloride adducts of oligosaccharides have gas-phase acidities lower than or close to that of HCl (1373 kJ/mol). In PSD experiments, precursor chloride adducts of oligosaccharides yield fragment ions that retain the charge on the sugar molecule rather than solely forming [Cl.sup.-], and these fragments can provide structurally informative product ions. In negative ion MALDI, highly acidic oligosaccharides do not form adducts with chloride anions, but mildly acidic saccharides (e.g., containing a carboxylic acid group) form both deprotonated molecules and chloride adducts, and each may provide structural information concerning the oligosaccharide upon decomposition.

Attachment of small anions to neutral molecules is an important ionization mechanism in negative ion electrospray mass spectrometry. In this report, the tendency for different anions to remain attached to selected analyte compound classes has been systematically investigated. A rationale for the formation and stability of preferred anionic adducts is proposed in light of thermodynamic considerations. A series of collision-induced dissociation experiments reveals that the gas-phase basicities of the deprotonated analyte molecule ([[M - H].sup.-]) and the anion moiety play important roles in determining the stability of anionic adducts. Adducts of the form [[M - H].sup.-] ... [H.sup.+] ... [[anion].sup.-] manifest increased stability when the two anions have similar gas-phase basicities. Within certain limitations, the difference in [DELTA] G [degrees] values for proton combination with [[M - H].sup.-] and with [[anion].sup.-] can be used as a first-order predictor of adduct stability. In addition, stability increases with the rising gas-phase basicities of the two moieties. The specific interaction between a small inorganic anion (bisulfate) and a neutral analyte molecule ([alpha]-D-glucose) in the form of multiple hydrogen bonding has also been affirmed by computer modeling to contribute to the stability of some anionic adducts. Last, the gas-phase basicity of deprotonated [alpha]-D-glucose (i.e., the gas-phase acidity of [alpha]-D-glucose) is determined by a 'bracketing method' to be in the range of 1373-1407 kJ/mol.

Brevetoxins, the toxic components of 'red tide' algae, all share one of two robust polycyclic ether backbone structures, but they are distinguished by differing side-chain substituents. Electrospray ionization mass spectrometry analyses of brevetoxins have shown that the polyether structure invariably has a very high affinity for sodium cations that results in the production of abundant (M + Na)(super +) ions even when sodium cations are only present as impurities. Because the ionic charge tends to remain localized on the sodium atom and because at least two bonds must be broken in order to produce polycyclic backbone fragmentation, it is extremely difficult to obtain abundant product ions (other than Na(super +)) from (M + Na)(super +) brevetoxin precursor ions in low-energy collision-induced dissociation (CID) MS/MS experiments. This report establishes that acid additives (oxalic acid, trifluoroacetic acid, and particularly hydrochloric acid) in aqueous methanol solutions can promote high yields of protonated brevetoxin molecules (MH(super +) ions) for Btx-1, -2, and -9 brevetoxins. Most importantly, unlike their (M + Na)(super +) counterparts, MH(super +) precursor ions offer readily detectable product ions in CID MS/MS experiments, even under low-energy collisions. This direct structural characterization approach has provided decomposition information from brevetoxins that was previously inaccessible, including the identification of diagnostic product ions for 'type A' brevetoxins (m/z 611) and 'type B' brevetoxins (m/z 779, 473, 179) and characteristic ions for Btx-1 (m/z 221, 139), Btx-2 (m/z 153), and Btx-9 (m/z 157, 85). Precursor ion scans and constant neutral loss scans are proposed to enable screening of individual type A or type B brevetoxins present in naturally occurring mixtures.

Lipid material represents a potential interference for determination of nonpolar compounds (e.g., polycyclic aromatic hydrocarbons) in biological tissue samples. This study reports the development of a selective extraction method using supercritical C[O.sub.2] that allows the GC/MS quantitation of PAHs in the presence of a substantial lipid background. Selective extraction of PAHs relies upon addition of [C.sub.18] adsorbent beads to the initial sample slurry. The dried mixture, including [C.sub.18] adsorbent, is placed in the supercritical fluid extraction (SFE) chamber. During the SFE process, lipids are preferentially retained on the [C.sub.18] beads. This 'SFE plus [C.sub.18]' procedure was developed by first optimizing SFE conditions (100 [degrees] C, 350 bar) for recovery of PAH standards. PAHs containing added model lipid compounds (stearic acid and cholesterol) were then subjected to SFE plus [C.sub.18] treatment followed by GC/MS analysis. Using this approach, a recovery of 94-100% of PAHs was obtained while only 9-17% of the lipid material present was coextracted from the same test sample. The developed method is demonstrated to permit efficient recovery and detection of PAHs spiked into crab tissue, a matrix with a high lipid content.

The ability to observe abundant gas-phase metal cluster ions in electrospray ionization mass spectrometry (ESI-MS) is highly dependent on experimental conditions. Alkali halides (MX) and other alkali metal salts were used to investigate the formation of cluster ions in ESI-MS. All compounds were found to give cluster ions of the form [([M.sub.n - 1][X.sub.n]).sup.+] and [([M.sub.n][X.sub.n -]).sup.-], with only two alkali salts yielding doubly charged cluster ions. In homologous alkali halide series, the relative abundances of cluster ions increased with increasing size of either the cation (positive ion mode) or the anion (negative ion mode). Calculations using an electrostatic model show that the gas-phase stability of cluster ions is greater for smaller cations or anions when a fixed counterion is employed. This stability calculation goes in a direction just opposite to the trend in cluster ion abundances observed in ESI-MS. Studies of equimolar mixtures consisting of two alkali halides reveal two distinct trends. When the equimolar mixture was composed of differing ions that participate in the droplet charge excess with the same counterion, the less solvated ions were found to form more abundant cluster ions. When the ions participating in the charge excess were fixed, the preferred counterion in observed clusters was the one that is more solvated in solution and forms more stable clusters in the gas phase. These observations can be rationalized by an extended form of the charged residue model where the weakly solvated ions that are part of the charge excess are preferentially enriched in offspring droplets during uneven fission. By contrast, transfer of a particular counterion located in the bulk of the droplets to the offspring droplets is not disfavored when this counterion is strongly solvated.

Micellar electrokinetic capillary chromatography is a useful technique in the separation and quantification of 2,4-D and atrazine levels. The technique relies on analyte partitioning between a micellar pseudostationary phase and aqueous solution. Additionally, the technique uses sodium dodecyl sulfate as the surfactant. An analysis of sediments from Lousiana's Lake Pontchartrain was conducted using the separation technique.

A three-electrode electrochemical cell coupled to an electrospray (ES) mass spectrometer has been developed and employed for on-line linear sweep voltammetry-ES mass spectrometry and electrochemical reaction studies. Generated in this type of electrochemistry/mass spectrometry (EC/MS) experiment are ion abundance vs potential profiles that provide molecular weight and structural information pertaining to intermediates and products of anodic oxidation of diphenyl sulfide, anodic pyridation of 9,10-diphenylanthracene, and reduction of nitrobenzene. Anodic oxidation of diphenyl sulfide in methylene chloride media at the platinum electrode yielded diphenyl surfoxide and pseudodimer sulfonium ion along with products of further oxidation. The obtained ion abundance vs potential profiles offer additional evidence to corroborate previously proposed reaction mechanisms for pseudodimer sulfonium ion generation from diphenyl sulfide, as well as pyridine addition to DPA radical cation also in methylene chloride at the platinum electrode. From the latter experiment, a newly found reaction product, [[DPA(py) - H].sup.+], is reported. Despite the inherent tendency of ES devices to induce oxidation processes in the positive ion mode, intermediates and products of nitrobenzene reduction in nonaqueous and organic/water media were detected in both negative and positive ES modes.

A newly invented probe accessory for fast electrochemistry/electrospray mass spectrometry (EC/ESMS) is presented and evaluated. The device features a low-volume, three-electrode electrochemical cell which has been designed with a minimum distance between the working electrode and the 'Taylor cone' inherent to the electrospray process. This configuration limits the time between electrochemical generation of ions and mass spectrometric analysis to an absolute minimum. A fused-silica layer insulates the microcylinder working electrode from the sample solution until immediately prior to the electrospray region, postponing electrode processes until the last moment. The same fused-silica layer insulates the working electrode from the surrounding auxiliary electrode, a stainless steel capillary that also serves as the electrospray capillary. The performance and capabilities of the novel electrochemistry/electrospray mass spectrometry system have been evaluated using polycyclic aromatic hydrocarbons (PAHs) as test analytes. In the positive ion EC/ESMS mode, oxidized forms (one-electron removal) of PAHs are produced in high yield. The ability to analyze reaction products appearing subsequent to the initial oxidation is also demonstrated.

The effect of analyte concentrations upon charge state distributions in electrospray ionization mass spectrometry (ESMS) was investigated using four compounds which showed doubly charged and singly charged positive ions (gramicidin S, 4,4[prime]-bipiperidine), multiply charged negative ions (Trypan Blue), or doubly charged and singly charged negative ions (eosin Y) in ES mass spectra. For each of the above compounds, the charge associated with analyte molecules was calculated to increase in solution with increasing concentration, yet the analyte charge state distributions in ES mass spectra were observed to monotonically shift toward lower values. In order to rationalize these observations, we introduce the ratio N/[N.sub.o], the ratio of the number of excess charges on all droplets produced by electrospray (N) to the total number of analyte molecules in all droplets ([N.sub.o]). For each compound, the N/[N.sub.o] value decreases with increasing analyte concentration. The lower N/[N.sub.o] value is proposed to be an underlying factor critical to explaining the desorption of a higher proportion of gas-phase ions of lower charge state at elevated analyte concentrations. We propose that the lower N/[N.sub.o] value is indicative of a decreased efficiency of analyte charging and an increased level of ion pairing of charged analyte molecules with available counterions. Furthermore, in comparing N/[N.sub.o] values and observed analyte charge state distributions as functions of increasing analyte concentration, the decreasing N/[N.sub.o] ratio predicts a much greater extent of shifting of analyte charge states toward lower values than was observed. This implies that the actual degree of ion pairing in droplets was considerably alleviated as compared to that indicated by the calculated N/[N.sub.o] values. This finding conforms to a description of the ES process wherein uneven fission of droplets occurs at the Rayleigh limit, thereby generating offspring droplets of higher charge to mass ratio, hence an augmented N/[N.sub.o] value.

On-line high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESMS) has been successfully applied to the separation and identification of brevetoxins associated with 'red tide' algae. Brevetoxins are toxic polyethers produced by the marine dinoflagellate Gymnodinium breve. They are responsible for fish kills, and they pose certain health risks to humans. The LC-MS method employs reversed-phase microbore HPLC on a [C.sub.18] column with a mobile phase consisting of 85:15 methanol/water, a flow rate of 8 [[micro]Liter]/min, and a postcolumn split ratio of 3:1 (UV absorbance detector/mass spectrometer). A brevetoxin culture sample was found to contain at least six components, including two well-separated peaks corresponding to the brevetoxins PbTx-2 and PbTx-1, as well as several unknown compounds, including one with a molecular mass of 899 Da (possibly an isomer of PbTx-9). The brevetoxin molecules exhibited a high tendency to bind to alkali cations in positive ion ESMS. For standard PbTx-9, PbTx-2, and PbTx-1 brevetoxins analyzed on our LC-MS system, the detection limits (employing mass spectrometer scans of 100 m/z units) were determined to be less than 600 fmol, 1 pmol, and 50 fmol, respectively (S/N = 3); the total analysis time was about 35 min.

The variability of ESMS charge state distributions was investigated for a number of analytes when the ionic strength of their solutions was altered over a wide range. Solution ionic strength was adjusted by adding varying amounts of electrolytes (N[H.sub.4]OAc, CsCl, or [(n-[C.sub.4][H.sub.9]).sub.4]N+Cl-) to protein solutions of myoglobin and lysozyme. Measured solution pH values were found to be nearly constant over the entire range of CsCl and [(n-[C.sub.4][H.sub.9]).sub.4]N+Cl- addition, and a slight increase in pH was observed upon N[H.sub.4]OAc addition. Analyte peak intensities in positive ion ESMS reproducibly decreased as the solution ionic strength increased. However, analyte charge state distributions remained quite constant despite 4 orders of magnitude variation in electrolyte concentration. In negative ion ESMS of phenolphthalein diphosphate (free acid form) and eosin Y, analyte charge state distributions also exhibited only minor variations while signal intensity decreased dramatically with increasing solution ionic strength upon addition of CsCl. These observations in positive and negative ion ESMS provide evidence that 'spectator' electrolytes present in solution have no significant effect upon the degree of multiple charging. The factors which determine the distribution of multiply charged analyte ions in the gas phase are thus independent of certain others which influence the efficiency of production of gas-phase analyte ions. In the context of theoretical descriptions of the electrospray process, the rate constants for desorption of multiply charged ions into the gas phase described in ion evaporation theory are likely to be invariant with spectator electrolyte concentration. Alternatively, the presence of added ion pairs in the ultimate droplet produced according to single ion in a droplet theory does not alter the level of charge attachment/charge separation of analyte molecules.

Metallocenes, substituted metallocenes, and organometallic salts containing metal-carbon bonds have been investigated by electrospray mass spectrometry (ESMS). Organometallic salts yielded stable cations in high abundances; metallocenes appeared in the oxidized form (i.e., as bis(cyclopentadienyl) metal cations, Cp2M+). Identifications were confirmed by inspection of the natural isotope distribution patterns. ESMS response vs concentration (log-log) plots showed a linear range between 10 to the -9 and 10 to the -5 M for both Cp2Fe and (Cp2Fe)+(PF6)-. For solutions of equivalent concentration, however, the salt form always yielded a higher abundance of Cp2Fe+. Neutral metallocenes are ionized by an electrochemical oxidation (electron-removal) mechanism at or near the electrospray (ES) capillary (needle). Increasing electrospray appearance potentials for Cp2M+ (M = metal) formation correlated with increasing half-wave potentials for oxidation (E(sub 1/2)(ox)) and increasing ionization energies (IE) in the series: decamethylferrocene, 1,1 prime-dimethylferrocene, ferrocene, formylferrocene, carboxylferrocene, acetylferrocene, 1,1 prime-diacetylferrocene, osmocene, and ruthenocene. For osmocene (relatively high E(sub 1/2)-(ox) and IE), ESMS conditions were established whereby protonation was competitive with oxidation, thus creating two pathways of ion formation. In methylene chloride solvent, chloride ion was incorporated into the inner sphere of certain metallocenes subjected to ESMS, forming Cp2RuCl+ and Cp2-OsCl+ complexes via nucleophilic addition. These latter Ru-(IV) and OS(IV) species also formed analogous gas-phase complexes with trifluoroacetate ion and hydroxide ion. Results have been rationalized on the basis of ion stability considerations.

Surface-induced dissociation (SID) has been used to produce daughter ion spectra of small protonated peptides generated by fast atom bombardment (FAB). The relative abundances of daughter ions depends critically upon the energy of the ion/surface collision. A wide array of decomposition processes may be observed using E(sub LAB) collision energies in the range 10-20 eV. At approximately 13-eV collision energy, the variety of decomposition processes is maximized for the small peptides studied; hence, maximum structural information may be deduced. Collisionally-activated dissociations (CAD) using argon gas and the identical protonated peptides could not produce as large an array of daughter ions in a constant condition experiment. An apparent contradiction is thereby posed because SID is known to produce a narrow distribution of ion internal energies relative to CAD. This apparent contradiction is resolved by considering the rather large kinetic energy spread of ions leaving the FAB source. For the SID process, this large initial kinetic energy distribution is converted into a significantly wider spread in center-of-mass collision energy, leading to a broader variety of decomposition processes (high and low energy) compared to CAD.

Abstract Background Combustion generated particulate matter is deposited in the respiratory tract and pose a hazard to the lungs through their potential to cause oxidative stress and inflammation. We have previously shown that combustion of fuels and chlorinated hydrocarbons produce semiquinone-type radicals that are stabilized on particle surfaces (i.e. environmentally persistent free radicals; EPFRs). Because the composition and properties of actual combustion-generated particles are complex, heterogeneous in origin, and vary from day-to-day, we have chosen to use surrogate particle systems. In particular, we have chosen to use the radical of 2-monochlorophenol (MCP230) as the EPFR because we have previously shown that it forms a EPFR on Cu(II)O surfaces and catalyzes formation of PCDD/F. To understand the physicochemical properties responsible for the adverse pulmonary effects of combustion by-products, we have exposed human bronchial epithelial cells (BEAS-2B) to MCP230 or the CuO/silica substrate. Our general hypothesis was that the EPFR-containing particle would have greater toxicity than the substrate species. Results Exposure of BEAS-2B cells to our combustion generated particle systems significantly increased reactive oxygen species (ROS) generation and decreased cellular antioxidants resulting in cell death. Resveratrol treatment reversed the decline in cellular glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels for both types of combustion-generated particle systems. Conclusion The enhanced cytotoxicity upon exposure to MCP230 correlated with its ability to generate more cellular oxidative stress and concurrently reduce the antioxidant defenses of the epithelial cells (i.e. reduced GSH, SOD activity, and GPx). The EPFRs in MCP230 also seem to be of greater biological concern due to their ability to induce lipid peroxidation. These results are consistent with the oxidizing nature of the CuO/silica ultrafine particles and the reducing nature and prolonged environmental and biological lifetimes of the EPFRs in MCP230.