High-mass cluster ions of ionic liquids in positive-ion and negative-ion DART-MS and their application for wide-range mass calibrations

Eight ionic liquids (ILs) are subjected to both positive-ion and negative-ion direct analyses in real time (DART) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). First, their ability to deliver evenly distributed cluster ion series covering a wide m/z range is explored. Then, one of the ILs exhibiting particularly useful cluster ion series in either ion polarity is applied for mass calibration. Using 1-butyl-3-methylimidazolium tricyanomethide delivers positive cluster ions suitable for mass calibration in the m/z 100–4,000 range and covers the m/z 100–2,000 range in negative-ion DART-MS. The corresponding mass reference lists are provided for either polarity. Furthermore, based on 1-butyl-3-methylimidazolium tricyanomethide, a high-mass record of m/z?>?5,000 for positive-ion DART-MS is presented. The mass calibration procedure is finally validated by application to established standard compounds such as polydimethylsiloxanes, perfluorononanoic acid, and Ultramark 1621, a mixture of hexakis (fluoroalkoxy) phosphazenes. Further proof is presented by consistent exact mass differences between adjacent cluster ions.

Inclusion Complexes of Ionic Liquids and Cyclodextrins: Are They Formed in the Gas Phase?

The interaction of imidazolium-based ionic liquids with α- and β-cyclodextrins was investigated by electrospray ionization mass spectrometry with variable collision induced dissociation energy and quantum chemical gas-phase calculations. The center-of-mass energy at which 50 % of a precursor ion decomposes (E_cm,1/2) was determined for the isolated [cyclodextrin + cation]⁺ or [cyclodextrin + anion]^– adduct ions of imidazolium-based ionic liquids with different alkyl chain lengths combined with a large set of anions, such as chloride, bromide, bis(trifluoromethylsulfonyl)imide, tetrafluoroborate, hexafluorophosphate, trifluoromethanesulfonate, methanesulfonate, dicyanamide, and hydrogensulfate. Moreover, both symmetric and asymmetric imidazolium cationic cores were evaluated. The relative interaction energies in the adduct ions were interpreted in terms of the influence of cation/anion structures and their inherent properties, such as hydrophobicity and hydrogen bond accepting ability, in the complexation process with the cyclodextrins. The trends observed in the mass spectral data together with quantum-chemical calculations suggest that in the gas phase, cations and anions will preferentially interact with the lower or upper rim of the cyclodextrin, respectively, as opposed to what has been reported in condensed phase where the formation of an inclusion complex between ionic liquid and cyclodextrin is assumed.

Reagent Cluster Anions for Multiple Gas-Phase Covalent Modifications of Peptide and Protein Cations

Multiple gas phase ion/ion covalent modifications of peptide and protein ions are demonstrated using cluster-type reagent anions of N-hydroxysulfosuccinimide acetate (sulfo-NHS acetate) and 2-formyl-benzenesulfonic acid (FBMSA). These reagents are used to selectively modify unprotonated primary amine functionalities of peptides and proteins. Multiple reactive reagent molecules can be present in a single cluster ion, which allows for multiple covalent modifications to be achieved in a single ion/ion encounter and at the ‘cost’ of only a single analyte charge. Multiple derivatizations are demonstrated when the number of available reactive sites on the analyte cation exceeds the number of reagent molecules in the anionic cluster (e.g., data shown here for reactions between the polypeptide [K₁₀ + 3H]³⁺ and the reagent cluster [5R_5Na – Na]^–). This type of gas-phase ion chemistry is also applicable to whole protein ions. Here, ubiquitin was successfully modified using an FBMSA cluster anion which, upon collisional activation, produced fragment ions with various numbers of modifications. Data for the pentamer cluster are included as illustrative of the results obtained for the clusters comprised of two to six reagent molecules.

Bioanalytical separation and preconcentration using ionic liquids

Ionic liquids (ILs) are novel solvents that display a number of unique properties, such as negligible vapor pressure, thermal stability (even at high temperatures), favorable viscosity, and miscibility with water and organic solvents. These properties make them attractive alternatives to environmentally unfriendly solvents that produce volatile organic compounds. In this article, a critical review of state-of-the-art developments in the use of ILs for the separation and preconcentration of bioanalytes in biological samples is presented. Special attention is paid to the determination of various organic and inorganic analytes—including contaminants (e.g., pesticides, nicotine, opioids, gold, arsenic, lead, etc.) and functional biomolecules (e.g., testosterone, vitamin B₁₂, hemoglobin)—in urine, blood, saliva, hair, and nail samples. A brief introduction to modern microextraction techniques based on ILs, such as dispersive liquid–liquid microextraction (DLLME) and single-drop microextraction (SDME), is provided. A comparison of IL-based methods in terms of their limits of detection and environmental compatibilities is also made. Finally, critical issues and challenges that have arisen from the use of ILs in separation and preconcentration techniques are also discussed.

Selective and Nonselective Cleavages in Positive and Negative CID of the Fragments Generated from In-Source Decay of Intact Proteins in MALDI-MS

Selective and nonselective cleavages in ion trap low-energy collision-induced dissociation (CID) experiments of the fragments generated from in-source decay (ISD) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of intact proteins are described in both positive and negative ion modes. The MALDI-ISD spectra of the proteins demonstrate common, discontinuous, abundant c- and z′-ions originating from cleavage at the N–Cα bond of Xxx-Asp/Asn and Gly-Xxx residues in both positive- and negative-ion modes. The positive ion CID of the c- and z′-ions resulted in product ions originating from selective cleavage at Asp-Xxx, Glu-Xxx and Cys-Xxx residues. Nonselective cleavage product ions rationalized by the mechanism of a “mobile proton” are also observed in positive ion CID spectra. Negative ion CID of the ISD fragments results in complex product ions accompanied by the loss of neutrals from b-, c-, and y-ions. The most characteristic feature of negative ion CID is selective cleavage of the peptide bonds of acidic residues, Xxx-Asp/Glu/Cys. A definite influence of α-helix on the CID product ions was not obtained. However, the results from positive ion and negative ion CID of the MALDI-ISD fragments that may have long α-helical domains suggest that acidic residues in helix-free regions tend to degrade more than those in helical regions.

Multiplex Mass Spectrometric Imaging with Polarity Switching for Concurrent Acquisition of Positive and Negative Ion Images

We have recently developed a multiplex mass spectrometry imaging (MSI) method which incorporates high mass resolution imaging and MS/MS and MS³ imaging of several compounds in a single data acquisition utilizing a hybrid linear ion trap-Orbitrap mass spectrometer (Perdian and Lee, Anal. Chem. 82, 9393–9400, 2010). Here we extend this capability to obtain positive and negative ion MS and MS/MS spectra in a single MS imaging experiment through polarity switching within spiral steps of each raster step. This methodology was demonstrated for the analysis of various lipid class compounds in a section of mouse brain. This allows for simultaneous imaging of compounds that are readily ionized in positive mode (e.g., phosphatidylcholines and sphingomyelins) and those that are readily ionized in negative mode (e.g., sulfatides, phosphatidylinositols and phosphatidylserines). MS/MS imaging was also performed for a few compounds in both positive and negative ion mode within the same experimental set-up. Insufficient stabilization time for the Orbitrap high voltage leads to slight deviations in observed masses, but these deviations are systematic and were easily corrected with a two-point calibration to background ions.

Fluorescence Spectroscopic Analysis of the Interaction of Papain with Ionic Liquids

The interaction between papain and two typical ionic liquids (ILs), 1-octyl-3-methylimidazolium chloride ([C₈mim]Cl) and 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl), was investigated by using fluorescence spectroscopy technique at a pH value of 7.4. The results suggested that ILs could quench the intrinsic fluorescence of papain probably via a static quenching mechanism. The binding constants were determined by employing the fluorescence quenching method. They were very small compared with that of volatile organic solvents, indicating that only very weak interaction between ILs and papain existed. The Gibbs free energy change (?G), enthalpy change (?H), and entropy change (?S) during the interaction of papain and ILs were estimated. Negative values of these parameters indicated that the interaction between ILs and papain was a spontaneous process, also implying that hydrogen bonding and van der Waals forces played important roles in the interaction processes.

Twelve Million Resolving Power on 4.7 T Fourier Transform Ion Cyclotron Resonance Instrument with Dynamically Harmonized Cell—Observation of Fine Structure in Peptide Mass Spectra

Resolving power of about 12,000 000 at m/z 675 has been achieved on low field homogeneity 4.7 T magnet using a dynamically harmonized Fourier transform ion cyclotron resonance (FT ICR) cell. Mass spectra of the fine structure of the isotopic distribution of a peptide were obtained and strong discrimination of small intensity peaks was observed in case of resonance excitation of the ions of the whole isotopic cluster to the same cyclotron radius. The absence of some peaks from the mass spectra of the fine structure was explained basing on results of computer simulations showing strong ion cloud interactions, which cause the coalescence of peaks with m/z close to that of the highest magnitude peak. The way to prevent peak discrimination is to excite ion clouds of different m/z to different cyclotron radii, which was demonstrated and investigated both experimentally and by computer simulations.

Elimination of Benzene from Protonated N-Benzylindoline: Benzyl Cation/Proton Transfer or Direct Proton Transfer?

Collision-induced dissociation (CID) of protonated N-benzylindoline and its derivatives was investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Elimination of benzene was observed besides hydride transfer and electron transfer reactions. D-labeling experiments and accurate mass determinations of the product ions confirm that the external proton is retained in the fragment ion, and the elimination reaction was proposed to be initiated by benzyl cation transfer rather than proton transfer. Benzyl cation transfer from the nitrogen atom to one of the sp²-hybridized carbon atoms in the indoline core is the key step, and subsequent proton transfer reaction leads to the elimination of benzene. Density functional theory (DFT)-based calculations were performed and the computational results also support the benzyl cation/proton transfer mechanism.

Ionic Liquid Matrix-Enhanced Secondary Ion Mass Spectrometry: The Role of Proton Transfer

Room temperature ionic liquids (ILs) are effective matrices in secondary ion mass spectrometry (SIMS) and matrix assisted laser desorption ionization (MALDI). In this paper, we examine the role of proton transfer in the mechanism of secondary ion enhancement using IL matrices in SIMS. We employ hydrogenated and deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as analytes to investigate the origin of proton transfer. The data indicate that protons from the IL anion transfer to the analyte in solution leading to an increase in the secondary ion intensity of the protonated molecular ion. The chemical identity of the matrix cation also affects analyte signal intensities. Using deuterated DPPC we observe that protons (deuterium) from the DPPC tail group react with the cation of the IL liquid leading to an increase in (cation + D)⁺ ion intensities. Further, the data suggest that the transfer kinetics of deuterium (hydrogen) is correlated with the secondary ion enhancements observed. The highest secondary ion enhancements are observed for the least sterically hindered cation. Neither the proton affinity nor the pK_a of the IL cation have a large effect on the analyte ion intensities, suggesting that steric factors are important in determining the efficacy of IL matrices for a given analyte.      

Soft Ionization of Saturated Hydrocarbons, Alcohols and Nonpolar Compounds by Negative-Ion Direct Analysis in Real-Time Mass Spectrometry

Large polarizable n-alkanes (approximately C18 and larger), alcohols, and other nonpolar compounds can be detected as negative ions when sample solutions are injected directly into the sampling orifice of the atmospheric pressure interface of the time-of-flight mass spectrometer with the direct analysis in real time (DART) ion source operating in negative-ion mode. The mass spectra are dominated by peaks corresponding to [M + O₂]￣^?. No fragmentation is observed, making this a very soft ionization technique for samples that are otherwise difficult to analyze by DART. Detection limits for cholesterol were determined to be in the low nanogram range.

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.

Compressive Mass Analysis on Quadrupole Ion Trap Systems

Conventionally, quadrupole ion trap mass spectrometers eject ions of different mass-to-charge ratio (m/z) in a sequential fashion by performing a scan of the rf trapping voltage amplitude. Due to the inherent sparsity of most mass spectra, the detector measures no signal for much of the scan time. By exploiting this sparsity property, we propose a new compressive and multiplexed mass analysis approach—multi Resonant Frequency Excitation (mRFE) ejection. This new approach divides the mass spectrum into several mass subranges and detects all the subrange spectra in parallel for increased mass analysis speed. Mathematical estimation of standard mass spectrum is demonstrated while statistical classification on the parallel measurements remains viable because of the sparse nature of the mass spectra. This method can reduce mass analysis time by a factor of 3–6 and increase system duty cycle by 2×. The combination of reduced analysis time and accurate compound classification is demonstrated in a commercial quadrupole ion trap (QIT) system.

CO2 laser ionization of acoustically levitated droplets

For many analytical purposes, direct laser ionization of liquids is desirable. Several studies on supported droplets, free liquid jets, and ballistically dispensed microdroplets have been conducted, yet detailed knowledge of the underlying mechanistics in ion formation is still missing. This contribution introduces a simple combination of IR-MALDI mass spectrometry and an acoustical levitation device for contactless confinement of the liquid sample. The homebuilt ultrasonic levitator supports droplets of several millimeters in diameter. These droplets are vaporized by a carbon dioxide laser in the vicinity of the atmospheric pressure interface of a time of flight mass spectrometer. The evaporation process is studied by high repetition rate shadowgraphy experiments elucidating the ballistic evaporation of the sample and revealing strong confinement of the vapor by the ultrasonic field of the trap. Finally, typical mass spectra for pure glycerol/water matrix and lysine as an analyte are presented with and without the addition of trifluoracetic acid, and the ionization mechanism is briefly discussed. The technique is a promising candidate for a reproducible mass spectrometric detection scheme for the field of microfluidics.

Thin layer chromatography–spray mass spectrometry: a method for easy identification of synthesis products and UV filters from TLC aluminum foils

A straightforward procedure for direct mass spectrometric (MS) analysis of spots from thin layer chromatography (TLC) plates, without the need of an external ion source, was developed using the aluminum plate backing as spray tip. The spots were cut out shaped as a tip with a 60° angle, mounted in front of the MS orifice, and after addition of a spray solvent spectra were obtained immediately. A high-resolution time-of-flight MS was used since the method is of particular interest for rapid identification or confirmation of spots from TLC plates. The practical benefits of this technique were demonstrated by detection of by-products of organic reactions, by identification of degradation products, and by accurate confirmation of spots when UV filters in sunscreens were analyzed by TLC. Employing the described method TLC spots can be evaluated fast without the need of an external ion source or devices for analyte transfer from TLC to MS, only a basic MS instrument and a high-voltage power supply is required.

Gas-Phase Fragmentations of Anions Derived from N-Phenyl Benzenesulfonamides

In addition to the well-known SO₂ loss, there are several additional fragmentation pathways that gas-phase anions derived from N-phenyl benzenesulfonamides and its derivatives undergo upon collisional activation. For example, N-phenyl benzenesulfonamide fragments to form an anilide anion (m/z 92) by a mechanism in which a hydrogen atom from the ortho position of the benzenesulfonamide moiety is specifically transferred to the charge center. Moreover, after the initial SO₂ elimination, the product ion formed undergoes primarily, an inter-annular H₂ loss to form a carbazolide anion (m/z 166) because the competing intra-annular H₂ loss is significantly less energetically favorable. Results from tandem mass spectrometric experiments conducted with deuterium-labeled compounds confirmed that the inter-ring mechanism is the preferred pathway. Furthermore, N-phenyl benzenesulfonamide and its derivatives also undergo a phenyl radical loss to form a radical ion with a mass-to-charge ratio of 155, which is in violation of the so-called “even-electron rule.”

Direct Identification of Tyrosine Sulfation by using Ultraviolet Photodissociation Mass Spectrometry

Sulfation is a common post-translational modification of tyrosine residues in eukaryotes; however, detection using traditional liquid chromatography-mass spectrometry (LC-MS) methods is challenging based on poor ionization efficiency in the positive ion mode and facile neutral loss upon collisional activation. In the present study, 193 nm ultraviolet photodissociation (UVPD) is applied to sulfopeptide anions to generate diagnostic sequence ions, which do not undergo appreciable neutral loss of sulfate even using higher energy photoirradiation parameters. At the same time, neutral loss of SO₃ is observed from the precursor and charge-reduced precursor ions, a spectral feature that is useful for differentiating tyrosine sulfation from the nominally isobaric tyrosine phosphorylation. LC-MS detection limits for UVPD analysis in the negative mode were determined to be around 100 fmol for three sulfated peptides, caerulein, cionin, and leu-enkephalin. The LC-UVPD-MS method was applied for analysis of bovine fibrinogen, and its key sulfated peptide was confidently identified.

The Collision Cross Sections of Iodide Salt Cluster Ions in Air via Differential Mobility Analysis-Mass Spectrometry

To date, most collision cross section (CCS) predictions have invoked gas molecule impingement-reemission rules in which specular and elastic scattering of spherical gas molecules from rigid polyatomic surfaces are assumed. Although such predictions have been shown to agree well with CCSs measured in helium bath gas, a number of studies reveal that these predictions do not agree with CCSs for ions in diatomic gases, namely, air and molecular nitrogen. To further examine the validity of specular-elastic versus diffuse-inelastic scattering models, we measured the CCSs of positively charged metal iodide cluster ions of the form [MI]_n[M⁺]_z, where M?=?Na, K, Rb, or Cs, n?=?1 – 25, and z?=?1 – 2. Measurements were made in air via differential mobility analysis mass spectrometry (DMA-MS). The CCSs measured are compared with specular-elastic as well as diffuse-inelastic scattering model predictions with candidate ion structures determined from density functional theory. It is found that predictions from diffuse-inelastic collision models agree well (within 5 %) with measurements from sodium iodide cluster ions, while specular-elastic collision model predictions are in better agreement with cesium iodide cluster ion measurements. The agreement with diffuse-inelastic and specular-elastic predictions decreases and increases, respectively, with increasing cation mass. However, even when diffuse-inelastic cluster ion predictions disagree with measurements, the disagreement is of a near-constant factor for all ions, indicating that a simple linear rescaling collapses predictions to measurements. Conversely, rescaling cannot be used to collapse specular-elastic predictions to measurements; hence, although the precise impingement reemission rules remain ambiguous, they are not specular-elastic.

Cyanide–Arene Meisenheimer Complex Generated in Electrospray Ionization Mass Spectrometry Using Acetonitrile as a Solvent

The C – C bond formation activated under negative electrospray ionization of an acetonitrile solution of 1,3,5-trinitrobenzene is reported. The solvent function is to provide a source of cyanide ion, a highly problematic reagent, which is found to attack the electron-deficient aromatic ring to form a covalently bound anionic complex (Meisenheimer complex). The structure of the complex is elucidated by means of collision induced dissociation mass spectrometry and IR multiple photon dissociation spectroscopy in the ‘fingerprint’ region.

Traveling Wave Ion Mobility Mass Spectrometry and Ab Initio Calculations of Phosphoric Acid Clusters

Positive and negative ion electrospray mass spectra obtained from 50 mM phosphoric acid solutions presented a large number of phosphoric acid clusters: [(H₃PO₄)_n?+?zH] ^z+ or [(H₃PO₄)_n – zH] ^z– , with n up to 200 and z up to 4 for positively charged clusters, and n up to 270 and z up to 7 for negatively charged cluster ions. Ion mobility experiments allowed very explicit separation of the different charge states. Because of the increased pressures involved in ion mobility experiments, dissociation to smaller clusters was observed both in the trap and transfer areas. Voltages along the ion path could be optimized so as to minimize this effect, which can be directly associated with the cleavage of hydrogen bonds. Having excluded the ion mobility times that resulted from dissociated ions, each cluster ion appeared at a single drift time. These drift times showed a linear progression with the number of phosphoric atoms for cluster ions of the same charge state. Cross section calculations were carried out with MOBCAL on DFT optimized geometries with different hydrogen locations and with three types of atomic charges. DFT geometry optimizations yielded roughly spherical structures. Our results for nitrogen gas interaction cross sections showed that values were dependent on the atomic charges definition used in the MOBCAL calculation. This pinpointed the necessity to define a clear theoretical framework before any comparative interpretations can be attempted with uncharacterized compounds.