Non-Direct Sequence Ions in the Tandem Mass Spectrometry of Protonated Peptide Amides—an Energy-Resolved Study

The fragmentation reactions of the MH⁺ ions of Leu-enkephalin amide and a variety of heptapeptide amides have been studied in detail as a function of collision energy using a QqToF beam type mass spectrometer. The initial fragmentation of the protonated amides involves primarily formation of b_n ions, including significant loss of NH₃ from the MH⁺ ions. Further fragmentation of these b_n ions occurs following macrocyclization/ring opening leading in many cases to b_n ions with permuted sequences and, thus, to formation of non-direct sequence ions. The importance of these non-direct sequence ions increases markedly with increasing collision energy, making peptide sequence determination difficult, if not impossible, at higher collision energies.

“Best Match” Model and Effect of Na+/H+ Exchange on Anion Attachment to Peptides and Stability of Formed Adducts in Negative Ion Electrospray Mass Spectrometry

The “Best Match” model has been extended to account for the role that Na⁺/H⁺ exchange plays on anion attachment in negative ion electrospray. Without any Na⁺/H⁺ exchange on (Glu) fibrinopeptide B, the higher basicity anions F^? and CH₃COO^? can hardly form observable adducts; however, after multiple Na⁺/H⁺ exchanges, adduct formation is enabled. Moreover, dissociation pathways of CF₃COO^? adducts with singly deprotonated peptides that have undergone 0 to 3 Na⁺/H⁺ exchanges exhibit a shift in CID product ions from losing predominately CF₃COOH (case of 0 Na⁺/H⁺ exchanges) to losing predominately CF₃COO^? (case of 3 Na⁺/H⁺ exchanges). These phenomena can be rationalized by considering that Na⁺ cations exchange at, and serve to “block”, the most acidic sites, thereby forcing implicated anions to attach to lower acidity protons. In addition to forming ion pairs with carboxylate groups, Na⁺ also participates in formation of tri-atomic ions of the form ANaA^? during adduct dissociation. The fact that low gas-phase basicity (GB) anions preferentially form ANaA^? species, even though high GB anions form more stable tri-atomic species, indicates that the monatomic ions were not in close contact in the initial adduct. The propensity for formation of stable anionic adducts is dependent on the degree of matching between anion GBs and GB_app of deprotonated sites on the peptide. The GB_app is raised dramatically as the charge state of the peptide increases via a through-space effect. The presence of Na⁺ on carboxylate sites substantially decreases the GB_app by neutralizing these sites, while slightly increasing the intrinsic GBs by an inductive effect.

Studying the Chemistry of Cationized Triacylglycerols Using Electrospray Ionization Mass Spectrometry and Density Functional Theory Computations

Analysis of triacylglycerols (TAGs), found as complex mixtures in living organisms, is typically accomplished using liquid chromatography, often coupled to mass spectrometry. TAGs, weak bases not protonated using electrospray ionization, are usually ionized by adduct formation with a cation, including those present in the solvent (e.g., Na⁺). There are relatively few reports on the binding of TAGs with cations or on the mechanisms by which cationized TAGs fragment. This work examines binding efficiencies, determined by mass spectrometry and computations, for the complexation of TAGs to a range of cations (Na⁺, Li⁺, K⁺, Ag⁺, NH₄ ⁺). While most cations bind to oxygen, Ag⁺ binding to unsaturation in the acid side chains is significant. The importance of dimer formation, [2TAG + M]⁺ was demonstrated using several different types of mass spectrometers. From breakdown curves, it became apparent that two or three acid side chains must be attached to glycerol for strong cationization. Possible mechanisms for fragmentation of lithiated TAGs were modeled by computations on tripropionylglycerol. Viable pathways were found for losses of neutral acids and lithium salts of acids from different positions on the glycerol moiety. Novel lactone structures were proposed for the loss of a neutral acid from one position of the glycerol moiety. These were studied further using triple-stage mass spectrometry (MS³). These lactones can account for all the major product ions in the MS³ spectra in both this work and the literature, which should allow for new insights into the challenging analytical methods needed for naturally occurring TAGs.

Cation-Induced Stabilization of Protein Complexes in the Gas Phase: Mechanistic Insights From Hemoglobin Dissociation Studies

Collision-induced dissociation (CID) of electrosprayed protein complexes usually involves asymmetric charge partitioning, where a single unfolded chain gets ejected that carries a disproportionately large fraction of charge. Using hemoglobin (Hb) tetramers as model system, we confirm earlier reports that bound metal ions can stabilize protein complexes under CID conditions. We examine the mechanism underlying this effect. Nonvolatile salts cause extensive adduct formation. Significant stabilization was observed for Mg²⁺ and Ca²⁺, whereas K⁺, Rb⁺, and Cs⁺ had no effect. Precursor ion selection was used to examine Hb subpopulations with well-defined metal binding levels. K⁺, Rb⁺, and Cs⁺-adducted tetramers eject monomers that carry roughly one-quarter of the metal ions that were bound to the precursor. This demonstrates that charge migration during CID is exclusively due to proton transfer, not metal ion transfer. Also, replacement of highly mobile charge carriers (protons) with less mobile species (metal ions) does not exert a stabilizing influence under the conditions used here. Interestingly, Hb carrying stabilizing ions (Mg²⁺ and Ca²⁺) generates monomeric CID products that are metal depleted. This effect is attributed to a combination of two factors: (1) Me²⁺ binding stabilizes Hb via formation of chelation bridges (e.g., R-COO^– Me^{2+ –}OOC-R); the more Me²⁺ a subunit contains the more stable it is. (2) More than ~90 % of the tetramers contain at least one subunit with a below-average number of Me²⁺. The prevalence of monomeric CID products with depleted Me²⁺ levels is caused by the tendency of these low metal-containing subunits to undergo preferential unfolding/ejection.

Controlling Dissociation Channels of Gas-Phase Protein Complexes Using Charge Manipulation

Coarse-grained simulations with charge hopping were performed for a positively charged tetrameric transthyretin (TTR) protein complex with a total charge of +20. Charges were allowed to move among basic amino acid sites as well as N-termini. Charge distributions and radii of gyration were calculated for complexes simulated at two temperatures, 300 and 600 K, under different scenarios. One scenario treated the complex in its normal state allowing charge to move to any basic site. Another scenario blocked protonation of all the N-termini except one. A final scenario used the complex in its normal state but added a basic-site containing tether (charge tag) near the N-terminus of one chain. The differences in monomer unfolding and charging were monitored in all three scenarios and compared. The simulation results show the importance of the N-terminus in leading the unfolding of the monomer units; a process that follows a zipper-like mechanism. Overall, experimentally modifying the complex by adding a tether or blocking the protonation of N-termini may give the potential for controlling the unraveling and subsequent dissociation of protein complexes.

Dissociations of Complexes Between Monovalent Metal Ions and Aromatic Amino Acid or Histidine

The fragmentations of [AA + M]⁺ complexes, where AA = Phe, Tyr, Trp, or His, and M is a monovalent metal (Li, Na, or Ag), have been exhaustively studied through collision-induced dissociation (CID) and through deuterium labeling. Dissociations of the Li- and Ag-containing complexes gave a large number of fragment ions; by contrast, the sodium/amino acid complexes have lower binding energies, and dissociation resulted in much simpler spectra, with loss of the entire ligand dominating. Unambiguous assignments of these fragment ions were made and formation mechanisms are proposed. Of particular interest are fragmentations in which the charge was retained on the organic fragment and the metal was lost, either as a metal hydride (AgH) or hydroxide (LiOH) or as the silver atom (Ag^?).

Native Electrospray Mass Spectrometry of DNA G-Quadruplexes in Potassium Solution

A commonly used electrolyte in electrospray mass spectrometry (ESI-MS) of biomolecules is ammonium acetate (NH₄OAc). Although some nucleic acid structures such as duplexes require only proper physiological ionic strength (whatever the monovalent ions) to be properly folded in ESI-MS conditions, the folding of some other nucleic acid structures such as DNA G-quadruplexes also depends on direct binding of specific cations. Here, we developed ESI-MS compatible conditions that allow one to observe DNA G-quaduplexes with K⁺ ions specifically bound between G-quartets. NH₄OAc was replaced with trimethylammonium acetate (TMAA), at concentrations up to 150 mM to provide physiological ionic strength, and the solution was doped with KCl at concentrations up to 1 mM. The trimethylammonium ion is too large to coordinate between G-quartets, where only K⁺ ions bind. Compared with the equivalent NH₄OAc/KCl mixtures, the TMAA/KCl mixtures provide cleaner spectra by suppressing the nonspecific adducts, and favor the formation of similar stacking arrangements as in 100 mM KCl (physiologically relevant cation) for the polymorphic human telomeric DNA G-quadruplexes. This new sample preparation method can be exploited to determine the number of potassium binding sites in new sequences, to screen ligand binding to the structures favored in potassium, and to transfer potassium-bound G-quadruplexes to the mass spectrometer for gas-phase structural probing, as illustrated herein with ion mobility spectrometry experiments.

Selective isolation of hemoglobin by use of imidazolium-modified polystyrene as extractant

Ionic liquids have attracted much attention in the analysis of a variety of species. The functional groups in ionic liquids can result in highly efficient separation and enrichment and, because of their typical lack of volatility, they are environmentally benign. We grafted imidazole cations onto the surface of chloromethyl polystyrene, denoted PS-CH₂-[MIM]⁺Cl^?, and this modified polymer was used to selectively extract the protein hemoglobin (Hb). The prepared extractant PS-CH₂-[MIM]⁺Cl^?, containing 2 mmol immobilized imidazole groups per gram polymer, was characterized by FT-IR, surface charge analysis, and elemental analysis. The adsorption efficiency was 91 %. The adsorption capacity of the PS-CH₂-[MIM]⁺Cl^? for Hb was 23.6 μg mg^?1, and 80 % of the retained Hb could be readily recovered by use of 0.5 % (m/v) aqueous sodium dodecyl sulfate (SDS) solution as eluate. The activity of the eluted Hb was approximately 90 %. The prepared imidazole-containing solid phase polymer was used for direct adsorption of Hb without use of any other solid matrix as support of the ionic liquid. The material was used in practice to isolate Hb from human whole blood.

Charge Site Mass Spectra: Conformation-Sensitive Components of the Electron Capture Dissociation Spectrum of a Protein

A conventional electron capture dissociation (ECD) spectrum of a protein is uniquely characteristic of the first dimension of its linear structure. This sequence information is indicated by summing the primary c ^m+ and z ^m+? products of cleavage at each of its molecular ion’s inter-residue bonds. For example, the ECD spectra of ubiquitin (M?+?nH)ⁿ⁺ ions, n?=?7–13, provide sequence characterization of 72 of its 75 cleavage sites from 1843 ions in seven c ^(1–7)+ and eight z ^(1–8)+? spectra and their respective complements. Now we find that each of these c/z spectra is itself composed of “charge site (CS)” spectra, the c ^m+ or z ^m+? products of electron capture at a specific protonated basic residue. This charge site has been H-bonded to multiple other residues, producing multiple precursor ion forms; ECD at these residues yields the multiple products of that CS spectrum. Closely similar CS spectra are often formed from a range of charge states of ubiquitin and KIX ions; this indicates a common secondary conformation, but not the conventional α-helicity postulated previously. CS spectra should provide new capabilities for comparing regional conformations of gaseous protein ions and delineating ECD fragmentation pathways.

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.

Influence of Metal–Peptide Complexation on Fragmentation and Inter-Fragment Hydrogen Migration in Electron Transfer Dissociation

The use of metal salts in electrospray ionization (ESI) of peptides increases the charge state of peptide ions, facilitating electron transfer dissociation (ETD) in tandem mass spectrometry. In the present study, K⁺ and Ca²⁺ were used as charge carriers to form multiply-charged metal–peptide complexes. ETD of the potassium- or calcium-peptide complex was initiated by transfer of an electron to a proton remote from the metal cation, and a c'-z? fragment complex, in which the c' and z? fragments were linked together via a metal cation coordinating with several amino acid residues, was formed. The presence of a metal cation in the precursor for ETD increased the lifetime of the c'-z? fragment complex, eventually generating c? and z' fragments through inter-fragment hydrogen migration. The degree of hydrogen migration was dependent on the location of the metal cation in the metal-peptide complex, but was not reconciled with conformation of the precursor ion obtained by molecular mechanics simulation. In contrast, the location of the metal cation in the intermediate suggested by the ETD spectrum was in agreement with the conformation of “proton-removed” precursors, indicating that the charge reduction of precursor ions by ETD induces conformational rearrangement during the fragmentation process.

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.

Role of Cationization and Multimers Formation for Diastereomers Differentiation by Ion Mobility-Mass Spectrometry

Stereochemistry plays an important role in biochemistry, particularly in therapeutic applications. Indeed, enantiomers have different biological activities, which can have important consequences. Many analytical techniques have been developed in order to allow the identification and the separation of stereoisomers. Here, we focused our work on the study of small diastereomers using the coupling of traveling wave ion mobility and mass spectrometry (TWIMS-MS) as a new alternative for stereochemistry study. In order to optimize the separation, the formation of adducts between diastereomers (M) and different alkali cations (X) was carried out. Thus, monomers [M + X]⁺ and multimers [2M + X]⁺ and [3M + X]⁺ ions have been studied from both experimental and theoretical viewpoints. Moreover, it has been shown that the study of the multimer [2Y + M + Li]⁺ ion, in which Y is an auxiliary diastereomeric ligand, allows the diastereomers separation. The combination of cationization, multimers ions formation, and IM-MS is a novel and powerful approach for the diastereomers identification. Thus, by this technique, diastereomers can be identified although they present very close conformations in gaseous phase. This work presents the first TWIMS-MS separation of diastereomers, which present very close collision cross section thanks to the formation of multimers and the use of an auxiliary diastereomeric ligand.

UV Photodissociation Action Spectroscopy of Haloanilinium Ions in a Linear Quadrupole Ion Trap Mass Spectrometer

UV–vis photodissociation action spectroscopy is becoming increasingly prevalent because of advances in, and commercial availability of, ion trapping technologies and tunable laser sources. This study outlines in detail an instrumental arrangement, combining a commercial ion-trap mass spectrometer and tunable nanosecond pulsed laser source, for performing fully automated photodissociation action spectroscopy on gas-phase ions. The components of the instrumentation are outlined, including the optical and electronic interfacing, in addition to the control software for automating the experiment and performing online analysis of the spectra. To demonstrate the utility of this ensemble, the photodissociation action spectra of 4-chloroanilinium, 4-bromoanilinium, and 4-iodoanilinium cations are presented and discussed. Multiple photoproducts are detected in each case and the photoproduct yields are followed as a function of laser wavelength. It is shown that the wavelength-dependent partitioning of the halide loss, H loss, and NH₃ loss channels can be broadly rationalized in terms of the relative carbon-halide bond dissociation energies and processes of energy redistribution. The photodissociation action spectrum of (phenyl)Ag₂ ⁺ is compared with a literature spectrum as a further benchmark.

Soft Supercharging of Biomolecular Ions in Electrospray Ionization Mass Spectrometry

The charge states of biomolecular ions in ESI-MS can be significantly increased by the addition of low-vapor supercharging (SC) reagents into the spraying solution. Despite the considerable interest from the community, the mechanistic aspects of SC are not well understood and are hotly debated. Arguments that denaturation accounts for the increased charging observed in proteins sprayed from aqueous solutions containing SC reagent have been published widely, but often with incomplete or ambiguous supporting data. In this work, we explored ESI MS charging and SC behavior of several biopolymers including proteins and DNA oligonucleotides. Analytes were ionized from 100 mM ammonium acetate (NH₄Ac) aqueous buffer in both positive (ESI+) and negative (ESI–) ion modes. SC was induced either with m-NBA or by the elevated temperature of ESI capillary. For all the analytes studied we, found striking differences in the ESI MS response to these two modes of activation. The data suggest that activation with m-NBA results in more extensive analyte charging with lower degree of denaturation. When working solution with m-NBA was analyzed at elevated temperatures, the SC effect from m-NBA was neutralized. Instead, the net SC effect was similar to the SC effect achieved by thermal activation only. Overall, our observations indicate that SC reagents enhance ESI charging of biomolecules via distinctly different mechanism compared with the traditional approaches based on analyte denaturation. Instead, the data support the hypothesis that the SC phenomenon involves a direct interaction between a biopolymer and SC reagent occurring in evaporating ESI droplets.

Evidence for Sequence Scrambling in Collision-Induced Dissociation of y-Type Fragment Ions

Sequence scrambling from y-type fragment ions has not been previously reported. In a study designed to probe structural variations among b-type fragment ions, it was noted that y fragment ions might also yield sequence-scrambled ions. In this study, we examined the possibility and extent of sequence-scrambled fragment ion generation from collision-induced dissociation (CID) of y-type ions from four peptides (all containing basic residues near the C-terminus) including: AAAAHAA-NH₂ (where “A” denotes carbon thirteen (¹³C₁) isotope on the alanine carbonyl group), des-acetylated-α-melanocyte (SYSMEHFRWGKPV-NH₂), angiotensin II antipeptide (EGVYVHPV), and glu-fibrinopeptide b (EGVNDNEEGFFSAR). We investigated fragmentation patterns of 32 y-type fragment ions, including y fragment ions with different charge states (+1 to +3) and sizes (3 to 12 amino acids). Sequence-scrambled fragment ions were observed from ~50 % (16 out of 32) of the studied y-type ions. However, observed sequence-scrambled ions had low relative intensities from ~0.1 % to a maximum of ~12 %. We present and discuss potential mechanisms for generation of sequence-scrambled fragment ions. To the best of our knowledge, results on y fragment dissociation presented here provide the first experimental evidence for generation of sequence-scrambled fragments from CID of y ions through intermediate cyclic “b-type” ions.

Charge Transfer Dissociation (CTD) Mass Spectrometry of Peptide Cations Using Kiloelectronvolt Helium Cations

A kiloelectronvolt beam of helium ions is used to ionize and fragment precursor peptide ions starting in the 1+ charge state. The electron affinity of helium cations (24.6 eV) exceeds the ionization potential of protonated peptides and can therefore be used to abstract an electron from—or charge exchange with—the isolated precursor ions. Kiloelectronvolt energies are used, (1) to overcome the Coulombic repulsion barrier between the cationic reactants, (2) to overcome ion-defocussing effects in the ion trap, and (3) to provide additional activation energy. Charge transfer dissociation (CTD) of the [M+H]⁺ precursor of Substance P gives product ions such as [M+H]^2+? and a dominant series of a ions in both the 1+ and 2+ charge states. These observations, along with the less-abundant a + 1 ions, are consistent with ultraviolet photodissociation (UVPD) results of others and indicate that C–C_α cleavages are possible through charge exchange with helium ions. Although the efficiencies and timescale of CTD are not yet suitable for on-line chromatography, this new approach to ion activation provides an additional potential tool for the interrogation of gas phase ions. Graphical Abstract

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Multiple stage pentaquadrupole mass spectrometry for generation and characterization of gas-phase ionic species. The case of the PyC2H 5 +· isomers

Eleven isomers with the PyC₂H ₅ ^+· composition, which include three conventional (1–3) and eight distonic radical cations (4–11), have been generated and in most cases successfully characterized in the gas phase via tandem-in-space multiple-stage pentaquadrupole MS² and MS³ experiments. The three conventional radical cations, that is, the ionized ethylpyridines C₂H₅-C₅H₄N^+· (1–3), were generated via direct 70-eV electron ionization of the neutrals, whereas sequences of chemical ionization and collision-induced dissociation (CID) or mass-selected ion-molecule reactions were used to generate the distonic ions H₂C^·?C₅H₄N⁺?CH₃ (4–6), CH₃?C₅H₄N⁺?CH ₂ ^· (7–9), C₅H₅N⁺?CH₂CH ₂ ^· (10), and C₅H₅N⁺?CH^·?CH₃ (11). Unique features of the low-energy (15-eV) CID and ion-molecule reaction chemistry with the diradical oxygen molecule of the isomers were used for their structural characterization. All the ion-molecule reaction products of a mass-selected ion, each associated with its corresponding CID fragments, were collected in a single three-dimensional mass spectrum. Ab initio calculations at the ROMP2/6–31G(d, p)//6–31G(d, p)+ZPE level of theory were performed to estimate the energetics involved in interconversions within the PyC₂H₅ ^+· system, which provided theoretical support for facile 4?7 interconversion evidenced in both CID and ion-molecule reaction experiments. The ab initio spin densities for the a-distonic ions 4–9 and 11 were found to be largely on the methylene or methyne formal radical sites, which thus ruled out substantial odd-spin derealization throughout the neighboring pyridine ring. However, only 8 and 9 (and 10) react extensively with oxygen by radical coupling, hence high spin densities on the radical site of the distonic ions do not necessarily lead to radical coupling reaction with oxygen. The very typical “spatially separated” ab initio charge and spin densities of 4–11 were used to classify them as distonic ions, whereas 1–3 show, as expected, “localized” electronic structures characteristic of conventional radical ions.     

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.

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.