Hydration of gas-phase ions formed by electrospray ionization

The hydration of gas-phase ions produced by electrospray ionization was investigated. Evidence that the hydrated ions are formed by two mechanisms is presented. First, solvent condensation during the expansion inside the electrospray source clearly occurs. Second, some solvent evaporation from more extensively solvated ions or droplets is apparent. To the extent that these highly solvated ions have solution-phase structures, then the final isolated gas-phase structure of the ion will be determined by the solvent evaporation process. This process was investigated for hydrated gramicidin S in a Fourier-transform mass spectrometer. Unimolecular dissociation rate constants of isolated gramicidin S ions with between 2 and 14 associated water molecules were measured. These rate constants increased from 16 to 230 s-1 with increasing hydration, with smaller values corresponding to magic numbers.     

Mononucleotide gas-phase proton affinities as determined by the kinetic method

The goal of this work is to determine the proton affinities of (deoxy)nucleoside 5'- and 3'-monophosphates (mononucleotides) using the kinetic method with fast atom bombardment mass spectrometry. The proton affinities of the (deoxy)nucleoside 5'- and 3'-monophosphates yielded the following trend: (deoxy)adenosine monophosphates > (deoxy)guanosine monophosphates > (deoxy)cytidine monophosphates > deoxythymidine/uridine monophosphates. In all cases the proton affinity decreases or remains the same with the addition of the phosphate group from those values reported for nucleosides. The proton affinity is dependent on the location of the phosphate backbone (5'-vs. 3'-phosphates): the 3'-monophosphates have lower proton affinities than the 5'-monophosphates except for the thymidine/uridine monophosphates where the trend is reversed. Molecular modeling was utilized to determine if multiple protonation sites and intramolecular hydrogen bond formation would influence the proton affinity measurements. Semiempirical calculations of the proton affinities at various locations on each mononucleotide were performed and compared to the experimental results. The possible influence of intramolecular hydrogen bonding between the nucleobases and the phosphate group on the measured and calculated proton affinities is discussed.     

Paper spray ionization of polar analytes using non-polar solvents

Non-polar solvents like hexane allow ionization of insoluble drugs, peptides, nucleotides and phospholipids as solids from paper. Ambient ionization is achieved simply by application of a high voltage to the wet paper. Transport and ionization mechanisms are discussed, including the possibility of field desorption from dendritic structures formed on the paper.     

Contribution of liquid-phase and gas-phase ionization in extractive electrospray ionization mass spectrometry of primary amines

In this study, we investigated how binary mixtures of compounds influence each other's signal intensity in electrospray ionization (ESI), extractive electrospray ionization (EESI) and secondary electrospray ionization (SESI) experiments. The experiments were conducted using a series of homologous primary amines (from 1-butyl to 1- decylamine). In every experiment, two of the amines were present, and all 21 possible combinations were measured with EESI, ESI and SESI as ionization sources. Except for the volatility, which decreases with increasing molecular weight, the physico-chemical properties of the amines are very similar, so that the intensity ratio obtained in each experiment provides information about discrimination effects occurring during the ionization process. The results show that for the relatively volatile compounds investigated, the EESI ionization mechanism resembles the SESI-like gas-phase charge transfer more than ESI-like analyte ionization in solution. In addition, almost no discrimination effects were observed in the spectra obtained in EESI experiments. Quantitative EESI experiments with nonylamine as internal standard showed that EESI is capable of providing both more accurate and more precise results than SESI and ESI.     

A new approach for the study of gas-phase ion-ion reactions using electrospray ionization

A simple flow reactor which facilitates the study and application of ion-ion and ion-molecule reactions at near atmospheric pressures is reported. Reactant ions were generated by electrospray ionization and discharge ionization methods, although any ionization sources amenable to atmospheric pressure may be used. Ions of opposite charge are generated in spatially separate ion sources and are swept into capillary inlets where the flows are merged and where reaction(s) can occur. Among the reactions investigated were the partial neutralization of multiply protonated polypeptides and proteins such as melittin, bradykinin, cytochrome c, and myoglobin by reaction with discharge-generated anions, the partial neutralization of multiply charged anions of oligodeoxyadenylic acid (d(pA)₃) by reaction with discharge-generated cations, the partial neutralization of bovine A-chain insulin anions by reaction with myoglobin [M+nH]ⁿ⁺ ions, and the reaction of multiply protonated melittin with discharge-generated cations. The cation-anion reactions generally resulted in a shift to lower charge (higher mass-to-charge ratio) in the products’ charge state distributions and the transfer of solvent molecules to the macromolecule products. Multiply protonated melittin was detected in a less highly solvated state with the positive discharge in operation.     

Polyglycine conformational analysis: calculated vs experimental gas-phase basicities and proton affinities

Structures of neutral and protonated polyglycines (Gly(n) and Gly(n)H(+) with n = 1-6) in the vicinity of global energy minima were calculated using the density functional theory at the B3LYP/6-311++G** (A) and B3LYP/6-31+G** (B) levels. Ninety-three structures were chosen for conformation and protonation studies. Geometries of the peptides are found to vary from open chains to multiple rings. Intramolecular hydrogen bonding is deduced to be the driving force for conformational stability. The preferred protonation sites are shown to be the terminal nitrogen atom and its adjacent amide oxygen atom. Structural series are developed according to geometrical form, hydrogen bonding, and protonation site. Physical factors that influence the relative electronic and thermodynamic stabilities of different structural series are examined. To obtain ab initio values of highest quality for gas-phase basicity (GB) and proton affinity (PA), electronic energies for n = 1-6 and thermal corrections to Gibbs free energy and enthalpy for n = 1-3 were calculated at level A, supplemented by thermal corrections for n = 4-6 at level B. Calculated GB and PA values are compared with mass spectral results obtained by the kinetic method (KM) and reaction bracketing (RB). The KM results and the ab initio values derived from structurally compatible pairs of lowest free energies are generally in good agreement, but the RB results for GB are lower by 2-8 kcal/mol for n = 2-6. Several reaction pathways are proposed to elucidate the experimental results. On the basis of theoretical structures consistent with the measurements, it is concluded that KM mostly samples the neutral and protonated structures of highest populations at thermal equilibrium, whereas RB targets those with sterically most accessible sites for protonation and deprotonation.     

Sweeping of neutral analytes in partial-filling micellar electrokinetic chromatography with electrospray ionization mass spectrometry

Sweeping improved the peak intensity of neutral analytes (i.e. dialkylphthalates) more than 100-fold without compromise to separation efficiency in partial-filling micellar electrokinetic chromatography with electrospray ionization mass spectrometry detection.     

Simultaneous sampling of volatile and non-volatile analytes in beer for fast fingerprinting by extractive electrospray ionization mass spectrometry

By gently bubbling nitrogen gas through beer, an effervescent beverage, both volatile and non-volatile compounds can be simultaneously sampled in the form of aerosol. This allows for fast (within seconds) fingerprinting by extractive electrospray ionization mass spectrometry (EESI-MS) in both negative and positive ion mode, without the need for any sample pre-treatment such as degassing and dilution. Trace analytes such as volatile esters (e.g., ethyl acetate and isoamyl acetate), free fatty acids (e.g., caproic acid, caprylic acid, and capric acid), semi/non-volatile organic/inorganic acids (e.g., lactic acid), and various amino acids, commonly present in beer at the low parts per million or at sub-ppm levels, were detected and identified based on tandem MS data. Furthermore, the appearance of solvent cluster ions in the mass spectra gives insight into the sampling and ionization mechanisms: aerosol droplets containing semi/non-volatile substances are thought to be generated via bubble bursting at the surface of the liquid; these neutral aerosol droplets then collide with the charged primary electrospray ionization droplets, followed by analyte extraction, desolvation, ionization, and MS detection. With principal component analysis, several beer samples were successfully differentiated. Therefore, the present study successfully extends the applicability of EESI-MS to the direct analysis of complex liquid samples with high gas content.

Sampling analytes from cheese products for fast detection using neutral desorption extractive electrospray ionization mass spectrometry

The development of analytical techniques suitable for sensitive, high-throughput, and nondestructive food analysis has been of increasing interest in recent years. In this study, mass-spectral fingerprints of various cheese products were rapidly recorded in the mass range of m/z 50–300 Da without any sample pretreatment, using neutral desorption extractive electrospray ionization mass spectrometry (ND-EESI-MS) in negative ion mode. The results demonstrate that both volatile and nonvolatile analytes on greasy cheese surfaces can be directly sampled by a neutral desorption gas beam. The influence of the neutral desorption gas flow on the analyte signal was systematically investigated. Under optimized experimental conditions, reproducible results were obtained using ND-EESI-MS. Principal component analysis was applied to differentiate a total of 49 individual cheese samples (four different types), which were purchased from three different supermarkets. All samples were successfully classified according to their types; but distributors and sensory properties were not distinguishable from the spectra data. The principal components 2, 3, and 4 scores showed an excellent capacity of distinguishing types of cheese. Molecular markers of interest can be identified using tandem mass spectrometry and matching the data with those from reference compounds. The experimental data show that ND-EESI-MS is able to sensitively and directly detect analytes on greasy surfaces without chemical contamination, providing a convenient method for high-throughput food analysis with a high degree of safety.     

Benchmark calculations of proton affinities and gas-phase basicities of molecules important in the study of biological phosphoryl transfer

Benchmark calculations of proton affinities and gas-phase basicities of molecules most relevant to biological phosphoryl transfer reactions are presented and compared with available experimental results. The accuracy of proton affinity and gas-phase basicity results obtained from several multi-level model chemistries (CBS-QB3, G3B3, and G3MP2B3) and density-functional quantum models (PBE0, B1B95, and B3LYP) are assessed and compared. From these data, a set of empirical bond enthalpy, entropy, and free energy corrections are introduced that considerably improve the accuracy and predictive capability of the methods. These corrections are applied to the prediction of proton affinity and gas-phase basicity values of important biological phosphates and phosphoranes for which experimental data does not currently exist. Comparison is made with results from semiempirical quantum models that are commonly employed in hybrid quantum mechanical/molecular mechanical simulations. Data suggest that the design of improved semiempirical quantum models with increased accuracy for relative proton affinity values is necessary to obtain quantitative accuracy for phosphoryl transfer reactions in solution, enzymes, and ribozymes.     

Evaluation of relative DNA binding affinities of anthrapyrazoles by electrospray ionization mass spectrometry

Binding interactions of a new series of anthrapyrazoles (APs) with DNA were evaluated by electrospray ionization mass spectrometry (ESI-MS). Relative binding affinities were estimated from the ESI-MS data based on the fraction of bound DNA for DNA/anthrapyrazole mixtures, and they show a correlation to the shift in melting point of the DNA measured from a previous study. Minimal sequence specificity was observed for the series of anthrapyrazoles. Upon collisionally activated dissociation of the duplex/anthrapyrazole complexes, typically ejection of the ligand was the dominant pathway for most of the complexes. However, for complexes containing AP2 or mitoxantrone, strand separation with the ligand remaining on one of the single strands was observed, indicative of a different binding mode or stronger binding.     

Uniformity of ionization response of structurally diverse analytes using a chip-based nanoelectrospray ionization source

The major drawback of liquid chromatography/mass spectrometry (LC/MS) for the analysis of mixtures is the non-quantitative nature of these studies. The ionization efficiency of the various components in the mixture (e.g., a compound and its metabolites) can vary greatly and, therefore, relative intensities of signals cannot be related to relative abundance. A chip-based nanoelectrospray ionization source was used to compare the ionization efficiencies of compounds with different physical-chemical characteristics. The data indicate that the ionization efficiencies vary much less with the chip-based device than by LC/MS. This was ascribed to the generation of a much higher electric field around the nozzles, which supplies a large excess of protons to the small droplets and reduces/eliminates the differences in the ionization efficiency for the analytes.     

Ab initio calculations and mass spectrometric determination of the gas-phase proton affinities of 4,4'-disubstituted 2,2'-bipyridines

The gas-phase proton affinities of 4,4'-di(R)-2,2'-bipyridines (R: H, Br, Cl, NO(2), Me) were determined by mass spectrometric measurements and by ab initio calculations at the HF/6-31G and MP2/6-31G levels of theory. The energy barriers for rotation about the central C-C bond were also studied computationally. Two minima were found for both unprotonated and protonated species, the global minima being at the trans planar and cis planar conformations, respectively. Local minima for the unprotonated compounds were at the cis nonplanar conformation and for the protonated compounds at the trans nonplanar. Two different proton affinity values were calculated for each compound by employing different conformations for the protonated species. The computational values were in good agreement with the experimental proton affinities. Substituents affect the proton affinity according to their ability to withdraw or to donate electrons, halogen and nitro-substituted bipyridines having a lower proton affinity and methyl-substituted bipyridine having a higher proton affinity than 2,2'-bipyridine itself.     

Influence of response factors on determining equilibrium association constants of non-covalent complexes by electrospray ionization mass spectrometry

A method for determining the equilibrium association constant of a complexation reaction A + B left harpoon over right harpoon AB by electrospray ionization mass spectrometry is described. The method consists in measuring the relative intensities of the peaks corresponding to A and to AB in equimolar A-B solutions at different concentrations C(0). The results are fitted by a non-linear least-squares procedure, with the two variable parameters being the equilibrium association constant K(a) and a factor R, defined by I(AB)/I(A) = R x [AB]/[A]. The factor R is the ratio between the response factors of AB and A, and corrects for the relative electrospray responses of the complex and the free substrate A, mass discrimination of instrumental origin and/or moderate in-source dissociation. The method is illustrated with the following two systems: complexes between a double-stranded 12-base pair oligonucleotide and minor groove binders, and cyclodextrin complexes with alpha,omega-dicarboxylic acids. For the oligonucleotide complexes, it is found that the response of the complex is not dramatically different to the response of the free oligonucleotide duplex, as the double helix conformation is disturbed by the drug only to a minor extent. In the case of cyclodextrin complexes, these complexes were found to have a much higher response than free cyclodextrin. This may be due to the fact that cyclodextrin is neutral in solution, whereas the complex is charged, but it can also stem from the fact that a significant proportion of the complex is in a non-inclusion geometry. The present method requires the exact determination of the concentrations of the reactants and is applicable to 1 : 1 complexes.     

Multilevel and density functional electronic structure calculations of proton affinities and gas-phase basicities involved in biological phosphoryl transfer

Five multilevel model chemistries (CBS-QB3, G3B3, G3MP2B3, MCG3/3, and MC-QCISD/3) and seven hybrid density functional methods (PBE0, B1B95, B3LYP, MPW1KCIS, PBE1KCIS, and MPW1B95) have been applied to the calculation of gas-phase basicity and proton affinity values for a series of 17 molecules relevant to the study of biological phosphoryl transfer. In addition, W1 calculations were performed on a subset of molecules. The accuracy of the methods was assessed and the nature of systematic errors was explored, leading to the introduction of a set of effective bond enthalpy and entropy correction terms. The multicoefficient correlation methods (MCG3/3 and MC-QCISD), with inclusion of specific zero-point scale factors, slightly outperform the other multilevel methods tested (CBS-QB3, G3B3, and G3MP2B3), with significantly less computational cost, and in the case of MC-QCISD, slightly less severe scaling. Four density functional methods, PBE1KCIS, MPW1B95, PBE0, and B1B95 perform nearly as well as the multilevel methods. These results provide an important set of benchmarks relevant to biological phosphoryl transfer reactions.     

Artifacts in amine analysis from anodic oxidation of organic solvents upon electrospray ionization for mass spectrometry

Different phenylenediamines were used to explore anodic oxidation in solution during electrospray ionization (ESI) mass spectrometry analysis. In our experiments, a series of unknown ionic species was detected in the phenylenediamine solutions. Our results propose that reactions of phenylenediamines with species formed by anodic oxidation of typical ESI solvents during the electrospray ionization process such as formaldehyde are producing these peaks. Identification of these compounds inter alia suggests formal alkylation, a reaction not reported so far as a result of electrolytic oxidation in the prospective organic solvents.     

Study of the electrospray ionization mass spectrometry of the proton pump inhibiting drug Omeprazole

A detailed analysis of the product ion spectrum generated from the protonated molecule under ESI-MS/MS conditions using a triple quadrupole mass spectrometer is reported for the gastrointestinal proton pump inhibitor Omeprazole. Unambiguous molecular composition data of the fragment ions were obtained with the aid of regioselectively 14C-, 34S- and 18O-labeled analogs. Attempts have been made to provide rational pathways for the formation of the fragment ions from four protonated omeprazole species. These results will facilitate the characterization of the complex metabolic fate of Omeprazole in humans, which involves the excretion of at least 50 metabolites.     

Relative importance of basicity in the gas phase and in solution for determining selectivity in electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is a critically important technique for the determination of small molecules, but its application for this purpose is complicated by its selectivity. For positive ion ESI-MS analysis of basic analytes, several investigators have pointed to the importance of analyte basicity as a source of selectivity. Currently, however, it is not known whether basicity in the gas phase or in solution is ultimately most important in determining responsiveness. The objective of these studies was to investigate the relative importance of basicity in solution and in the gas phase as factors that predict selectivity in positive ion ESI-MS analysis. ESI-MS response was compared for a diverse series of protonatable analytes in two different solvents, neat methanol and methanol with 0.5% acetic acid. A correlation was observed between analyte pK(b) and electrospray response. However, the response for the analytes with very high pK(b) values was significantly higher than would be expected based on concentration of the protonated form or the analyte in solution, and this higher response did not appear to result from gas-phase proton transfer reactions. Although all of the analytes investigated had higher gas-phase basicities than the solvent, their relative responses were not dictated by gas-phase basicity. Higher response was observed for all of the analytes studied in acidified methanol compared with neat methanol, and this higher response was most pronounced for weakly basic analytes. These findings support the use of analyte pK(b) for rational method development in ESI-MS analysis of small molecules.