Protonation-Induced Chirality Drives Separation by Differential Ion Mobility Spectrometry.

Upon development of a workflow to analyze (±)-Verapamil and its metabolites using differential mobility spectrometry (DMS), we noticed that the ionogram of protonated Verapamil consisted of two peaks. This was inconsistent with its metabolites, as each exhibited only a single peak in the respective ionograms. The unique behaviour of Verapamil was attributed to protonation at its tertiary amino moiety, which generated a stereogenic quaternary amine. The introduction of additional chirality upon N-protonation of Verapamil renders four possible stereochemical configurations for the protonated ion: (R,R), (S,S), (R,S), or (S,R). The (R,R)/(S,S) and (R,S)/(S,R) enantiomeric pairs are diastereomeric and thus exhibit unique conformations that are resolvable by linear and differential ion mobility techniques. Protonation-induced chirality appears to be a general phenomenon, as N-protonation of 12 additional chiral amines generated diastereomers that were readily resolved by DMS.

Observation of charged droplets from electrospray ionization (ESI) plumes in API mass spectrometers.

Electrospray ionization (ESI) generates bare analyte ions from charged droplets, which result from spraying a liquid in a strong electric field. Experimental observations available in the literature suggest that at least a significant fraction of the initially generated droplets remain large, have long lifetimes, and can thus aspirate into the inlet system of an atmospheric pressure ionization mass spectrometer (API-MS). We report on the observation of fragment signatures from charged droplets penetrating deeply the vacuum stages of three commercial mass spectrometer systems with largely different ion source and spray configurations. Charged droplets can pass through the ion source and pressure reduction stages and even into the mass analyzer region. Since droplet signatures were found in all investigated instruments, the incorporation of charged droplets is considered a general phenomenon occurring with common spray conditions in ESI sources.

Electronic spectroscopy of differential mobility-selected prototropic isomers of protonated para-aminobenzoic acid.

para-Aminobenzoic acid (PABA) was electrosprayed from mixtures of protic and aprotic solvents, leading to formation of two prototropic isomers in the gas phase whose relative populations depended on the composition of the electrospray solvent. The two ion populations were separated in the gas phase using differential mobility spectrometry (DMS) within a nitrogen-only environment at atmospheric pressure. Under high-field conditions, the two prototropic isomers eluted with baseline signal separation with the N-protonated isomer having a more negative CV shift than the O-protonated isomer, in accord with previous DMS studies. The conditions most favorable for formation and separation of each tautomer were used to trap each prototropic isomer in a quadrupole ion trap for photodissociation action spectroscopy experiments. Spectral interrogation of each prototropic isomer in the UV region (3-6 eV) showed good agreement with previously recorded spectra, although a previously reported band (4.8-5.4 eV) was less intense for the O-protonated isomer in our measured spectrum. Without DMS selection, the measured spectra contained features corresponding to both protonated isomers even when solvent conditions were optimised for formation of a single isomer. Interconversion between protonated isomers within the ion trap was observed when protic ESI solvents were employed, leading to spectral cross contamination even with mobility selection. CCSD vertical excitation energies and vertical gradient (VG) Franck-Condon simulations are presented and reproduce the measured spectral features with near-quantitative agreement, providing supporting evidence for spectral assignments.

UVPD spectroscopy of differential mobility-selected prototropic isomers of protonated adenine.

Two prototropic isomers of adenine are formed in an electrospray ion source and are resolved spatially in a differential mobility spectrometer before detection in a triple quadrupole mass spectrometer. Each isomer is gated in CV space before being trapped in the linear ion trap of the modified mass spectrometer, where they are irradiated by the tuneable output of an optical parametric oscillator and undergo photodissociation to form charged fragments with m/z 119, 109, and 94. The photon-normalised intensity of each fragmentation channel is measured and the action spectra for each DMS-gated tautomer are obtained. Our analysis of the action spectra, aided by calculated vibronic spectra and thermochemical data, allow us to assign the two signals in our measured ionograms to specific tautomers of protonated adenine.

UVPD Spectroscopy of Differential Mobility-Selected Prototropic Isomers of Rivaroxaban.

Two ion populations of protonated Rivaroxaban, [C19H18ClN3O5S + H]+, are separated under pure N2 conditions using differential mobility spectrometry prior to characterization in a hybrid triple quadrupole linear ion trap mass spectrometer. These populations are attributed to bare protonated Rivaroxaban and to a proton-bound Rivaroxaban-ammonia complex, which dissociates prior to mass-selecting the parent ion. Ultraviolet photodissociation (UVPD) and collision-induced dissociation (CID) studies indicate that both protonated Rivaroxaban ion populations are comprised of the computed global minimum prototropic isomer. Two ion populations are also observed when the collision environment is modified with 1.5% (v/v) acetonitrile. In this case, the protonated Rivaroxaban ion populations are produced by the dissociation of the ammonium complex and by the dissociation of a proton-bound Rivaroxaban-acetonitrile complex prior to mass selection. Again, both populations exhibit a similar CID behavior; however, UVPD spectra indicate that the two ion populations are associated with different prototropic isomers. The experimentally acquired spectra are compared with computed spectra and are assigned to two prototropic isomers that exhibit proton sharing between distal oxygen centers.

Enhancing signal and mitigating up-front peptide fragmentation using controlled clustering by gas-phase modifiers.

Up-front CID fragmentation is a phenomenon where molecular ions are activated and fragment as they enter the atmosphere-to-vacuum region of the mass spectrometer, and consequently can complicate the mass spectra and their analysis. This phenomenon can be minimized by controlling the voltages on lens/optic elements where ions are sampled from the atmospheric region, but this approach can also have a negative effect on overall ion sensitivity. In this study, we introduce gas-phase modifiers (acetonitrile, acetone, cyclohexane, water, and methanol) to the curtain gas to mitigate up-front CID fragmentation. These modifiers cluster with incoming ions, increasing the energy barrier to fragmentation and consequently reducing the complexity of mass spectra. The clustering is monitored by differential mobility spectrometry-mass spectrometry (DMS-MS) and precursor mass spectrum-scanning. Unlike typical singly charged species, peptide ion-modifier clusters were found to survive through the atmosphere-to-vacuum interface of the mass spectrometer, showing that highly charged peptides cluster most strongly with acetonitrile and acetone. In addition, when peptides cluster with acetonitrile, they produce a large increase in signal intensity for the most highly charged and fragile ions. This results in a significant reduction, up to 90% with some modifiers, in up-front CID fragmentation for these fragile highly charged peptides, increasing the overall analytical sensitivity and decreasing the limits of detection by up to 82% depending on the analyte. The proposed technique has no significant detrimental effect on the peptide mass fingerprinting of a BSA or mAb protein digest, but it does reduce the amount of redundant and data-deficient spectra needed to produce adequate sequence coverage using information-dependent acquisition methods by ~ 40%. We propose that this technique could have a benefit in the fields of proteomics and peptidomics where up-front CID fragmentation and chemical noise routinely mask targets of biological importance. Graphical abstract.

Differential mobility spectrometry/mass spectrometry history, theory, design optimization, simulations, and applications.

This review of differential mobility spectrometry focuses primarily on mass spectrometry coupling, starting with the history of the development of this technique in the Soviet Union. Fundamental principles of the separation process are covered, in addition to efforts related to design optimization and advancements in computer simulations. The flexibility of differential mobility spectrometry design features is explored in detail, particularly with regards to separation capability, speed, and ion transmission. 2015 Wiley Periodicals, Inc. Mass Spec Rev 35:687-737, 2016.

Polymer micronozzle array for multiple electrosprays produced by templated synthesis and etching of microstructured fibers.

Microstructured fibers (MSFs) having raised polymer nozzles in each channel are custom designed, fabricated, and tested for use as multiple electrospray (MES) emitters for mass spectrometry (MS). There is strong motivation to develop electrospray emitters that operate at practical flow rates but give the much greater ionization efficiency associated with lower (nano) flow rates. This can be accomplished by splitting the flow into many lower-volume electrosprays, an approach known as MES. To couple with most modern mass spectrometers, the MES emitter must have a small diameter to allow efficient ion collection into the MS. In this work, a MSF, defined as a fiber having many empty channels running along its length, was designed to have 9 channels, 9 µm each, >100 µm apart arranged in a radial pattern, all in a fiber having a compatible diameter with both front-end LC equipment and typical MS inlets. This design seeks to promote independent electrospray from each channel while maintaining electric field homogeneity. While the MSFs themselves do not support MES, the formation of polymer nozzles protruding from each channel at the tip face enables independent electrospray from each nozzle. Microscope imaging, electrospray current measurement, and ESI-MS detection of a model analyte all confirm the MES behavior of the 9-nozzle emitter, showing significant signal enhancement relative to a single-nozzle emitter at the same total flow rate. LC/MS data from a protein digest obtained at an independent laboratory demonstrates the applicability and robustness of the emitter for real scientific challenges using modern LC/MS equipment.

Open Port Interface for Coupling Capillary Electrophoresis and Mass Spectrometry: Performance Evaluation for Capillary Isoelectric Focusing.

Capillary electrophoresis (CE) combined with mass spectrometry (MS) is a powerful analytical technique that utilizes the resolving power of CE and the mass-detection capabilities of MS. In many cases, CE is coupled to MS via a sheath-flow interface (SFI). This interface has a simple design and can be easily constructed; however, it often suffers from issues such as MS signal suppression, interference of MS and CE electrical circuits, and the inability to set an optical point of detection close to the capillary end due to the specific design of the coupling union. In this paper, we describe a novel coupling of CE and MS based upon the open port interface (OPI). The OPI differs from classical sheath flow interfaces by operating at flow rates at least 1 order of magnitude higher. In addition to the flow rate difference, the OPI provides more efficient mixing of the capillary eluates with the transport fluid and thus minimizes MS signal suppression. In this work, we compared the performance of OPI and SFI in a series of capillary isoelectric focusing (cIEF) experiments with 5 pI markers, carbonic anhydrase II and NIST antibody. The evaluation criteria for the comparison of the OPI and SFI were analytical sensitivity, reproducibility, and pI marker linearity. Given the extent of sample dilution in the OPI, we also compared the peak resolution determined using an upstream UV detector to those determined by the downstream mass spectrometer. The results suggested that the OPI configuration reduced signal suppression, with no adverse effect on peak resolution. In addition, the OPI provided better decoupling of the CE and MS potentials as well as reduced signal dependence upon the sheath liquid composition. While these results are preliminary, they suggest that the OPI is a viable approach for CE-MS coupling.

Probing electrospray ionization dynamics using differential mobility spectrometry: the curious case of 4-aminobenzoic acid.

Here, we present the separation of two ions that differ only by the site of protonation of the analyte molecule using differential mobility spectrometry (DMS). Protonated 4-aminobenzoic acid molecules (4-ABA) generated by positive-mode electrospray ionization [ESI(+)] can exist with the proton residing on either the amine nitrogen (N-protonated) or the carboxylic acid oxygen (O-protonated), and the protonation site can differ on the basis of the solvent system used. In this study, we demonstrate the identification and separation of N- and O-protonated 4-ABA using DMS, with structural assignments verified by: (1) the presence of distinct peaks in the DMS ionogram, (2) the observed effects resulting from altering the ESI(+) solvent system, (3) the observed (13)C NMR chemical shifts arising from altering the solvent system, (4) the observation of distinct MS/MS fragmentation patterns for the two DMS-separated ions, (5) the unique hydrogen-deuterium exchange behavior for these ions, and (6) the fundamental behavior of these two ions within the DMS cell, linked back to the structural differences between the two protonated forms.

Differential mobility spectrometry of isomeric protonated dipeptides: modifier and field effects on ion mobility and stability.

The ability to resolve isomeric protonated dipeptides was investigated with the new technique of differential ion mobility mass spectrometry that uses "modifier" molecules to enhance differential mobility. Two pairs of protonated peptides [glycine-alanine (GlyAla) and alanine-glycine (AlaGly), glycine-serine (GlySer) and serine-glycine (SerGly)] and eight different modifiers (water, 2-propanol, 1,5-hexadiene, 2-chloropropane, chlorobenzene, dichloromethane, acetonitrile, and cyclohexane) were used in the initial study. Separation of the protonated peptides was found to be dependent on the mass and proton affinity of the modifier and combinations of functionalities present in the modifier and the analyte ion. Six of the eight modifiers (water, 2-propanol, chlorobenzene, cyclohexane, dichloromethane, and acetonitrile) were able to separate the protonated isomeric peptide pairs, and generally, modifiers with electron-rich groups performed the best. In the presence of some modifiers, a reduction of ion current was observed under the highest field conditions (>115 Td). Dopant-catalyzed isomerization, likely by proton-transport catalysis, and field-induced fragmentation may have contributed to these losses. Two high vapor pressure modifiers, 1,5-hexadiene and 2-chloropropane, significantly influenced ion formation leading to the formation of stable cluster populations that could be observed in the mass spectrometer. Although not a major concern, both fragmentation and influence of modifier evaporation warrant further studies in order to fully understand and possibly eliminate them.

Rapid analysis of isomeric exogenous metabolites by differential mobility spectrometry-mass spectrometry.

The direct separation of isomeric glucuronide metabolites from propranolol dosed tissue extracts by differential mobility spectrometry-mass spectrometry (DMS-MS) with the use of the polar gas-phase chemical modifier acetonitrile was demonstrated. The DMS gas-phase separation was able to resolve the isomeric metabolites with separation times on the order of milliseconds instead of minutes which is typically required when using pre-ionization chromatographic separation methods. Direct separation of isomeric metabolites from the complex tissue extract was confirmed by implementing a high-performance liquid chromatography (HPLC) separation prior to the DMS-MS analysis to pre-separate the species of interest. The ability to separate isomeric exogenous metabolites directly from a complex tissue extract is expected to facilitate the drug development process by increasing analytical throughput without the requirement for pre-ionization cleanup or separation strategies.

Ion Guide for Improved Atmosphere to Mass Spectrometer Vacuum Ion Transfer.

Various approaches for transmitting ions from atmosphere to the deep vacuum required for mass analysis have been developed with the goal to increase the ion to gas ratio while maintaining high ion transmission efficiency. Since the vast majority of ion losses occurs in the atmospheric pressure ion source, an effective way to improve sampling of those ions is to increase the atmosphere to vacuum aperture diameter. However, as the aperture diameter is increased, the resulting intense free jet gas expansion and subsequent gas beam can scatter ions in the first vacuum region. The interface described here provides an optimized flow field to the second vacuum stage, with a unique geometry to counter the ion losses from scattering collisions with the gas. Two additional differentially pumped quadrupole ion guides are used to further improve the ion to gas ratio, resulting in an ion transfer efficiency improvement of 5-6× over a two-stage differentially pumped interface with quadrupole ion guides. The interface also demonstrates efficient declustering and fragmentation capabilities beneficial for reducing background chemical noise.

Sampling Efficiency Improvement to an Electrospray Ionization Mass Spectrometer and Its Implications for Liquid Chromatography Based Inlet Systems in the Nanoliter to Milliliter per Minute Flow Range.

This paper describes electrospray sampling efficiency measurements obtained on a triple quadrupole mass spectrometer equipped with a large atmosphere to vacuum sampling aperture and modified ion optics designed to confine the ions traveling in the intense expanding gas beam and prevent scattering losses in the entrance optics of the mass analyzer. Sampling efficiency, defined as the ratio of the number of ions captured in the first vacuum stage of the entrance optics to the number of analyte molecules entering the ion source, is a measure of sensitivity that takes into account both ionization efficiency at atmospheric pressure, the efficiency of transporting the ions from atmosphere to vacuum, and the efficiency of confining them in the subsequent gas expansion before mass analysis. Sampling efficiency measurements were conducted under high-performance liquid chromatography sample introduction conditions using columns and flow rates spanning the nanoflow (300 nL/min), microflow (3-60 µL/min), and milliflow (100-500 µL/min) ranges. The results show a convergence in the sampling efficiencies across this range, narrowing the sensitivity gap between the nanoflow and higher flow rate ranges largely because nanoflow sampling efficiency has been shown to be close to 100% for more than a decade, leaving little room for improvement. Under situations where sample volumes are not limiting, lower concentration detection limits can now be achieved with the higher flow rate systems versus nanoflow as a direct consequence of the higher sample loading capacity of the columns and the reduction in the difference in their ion sampling efficiencies.

The Charge-State and Structural Stability of Peptides Conferred by Microsolvating Environments in Differential Mobility Spectrometry.

The presence of solvent vapor in a differential mobility spectrometry (DMS) cell creates a microsolvating environment that can mitigate complications associated with field-induced heating. In the case of peptides, the microsolvation of protonation sites results in a stabilization of charge density through localized solvent clustering, sheltering the ion from collisional activation. Seeding the DMS carrier gas (N2) with a solvent vapor prevented nearly all field-induced fragmentation of the protonated peptides GGG, AAA, and the Lys-rich Polybia-MP1 (IDWKKLLDAAKQIL-NH2). Modeling the microsolvation propensity of protonated n-propylamine [PrNH3]+, a mimic of the Lys side chain and N-terminus, with common gas-phase modifiers (H2O, MeOH, EtOH, iPrOH, acetone, and MeCN) confirms that all solvent molecules form stable clusters at the site of protonation. Moreover, modeling populations of microsolvated clusters indicates that species containing protonated amine moieties exist as microsolvated species with one to six solvent ligands at all effective ion temperatures (Teff) accessible during a DMS experiment (ca. 375-600 K). Calculated Teff of protonated GGG, AAA, and Polybia-MPI using a modified two-temperature theory approach were up to 86 K cooler in DMS environments seeded with solvent vapor compared to pure N2 environments. Stabilizing effects were largely driven by an increase in the ion's apparent collision cross section and by evaporative cooling processes induced by the dynamic evaporation/condensation cycles incurred in the presence of an oscillating electric separation field. When the microsolvating partner was a protic solvent, abstraction of a proton from [MP1 + 3H]3+ to yield [MP1 + 2H]2+ was observed. This result was attributed to the proclivity of protic solvents to form hydrogen-bond networks with enhanced gas-phase basicity. Collectively, microsolvation provides analytes with a solvent "air bag," whereby charge reduction and microsolvation-induced stabilization were shown to shelter peptides from the fragmentation induced by field heating and may play a role in preserving native-like ion configurations.

UV-induced bond modifications in thymine and thymine dideoxynucleotide: structural elucidation of isomers by differential mobility mass spectrometry.

Differential mobility spectrometry has been applied to reveal the occurrence of isomerization of thymine nucleobase and of thymine dideoxynucleotide d(5'-TT-3') due to bond redisposition induced by UV irradiation at 254 nm of frozen aqueous solutions of these molecules. Collision-induced dissociation (CID) spectra of electrosprayed photoproducts of the thymine solution suggest the presence of two isomers (the so-called cyclobutane and 6,4-photoproducts) in addition to the proton-bound thymine dimer, and these were separated using differential mobility spectrometry/mass spectrometry (DMS/MS) techniques with water as the modifier. Similar experiments with d(5'-TT-3') revealed the formation of a new isomer of deprotonated thymine dideoxynucleotide upon UV irradiation that was easily distinguished using DMS/MS with isopropanol as the modifier. The results reinforce the usefulness of DMS/MS in isomer separation.

Chemical effects in the separation process of a differential mobility/mass spectrometer system.

In differential mobility spectrometry (also referred to as high-field asymmetric waveform ion mobility spectrometry), ions are separated on the basis of the difference in their mobility under high and low electric fields. The addition of polar modifiers to the gas transporting the ions through a differential mobility spectrometer enhances the formation of clusters in a field-dependent way and thus amplifies the high- and low-field mobility difference, resulting in increased peak capacity and separation power. Observations of the increase in mobility field dependence are consistent with a cluster formation model, also referred to as the dynamic cluster-decluster model. The uniqueness of chemical interactions that occur between an ion and cluster-forming neutrals increases the selectivity of the separation, and the depression of low-field mobility relative to high-field mobility increases the compensation voltage and peak capacity. The effect of a polar modifier on the peak capacity across a broad range of chemicals has been investigated. We discuss the theoretical underpinnings which explain the observed effects. In contrast to the result with a polar modifier, we find that using mixtures of inert gases as the transport gas improves the resolution by reducing the peak width but has very little effect on the peak capacity or selectivity. The inert gas helium does not cluster and thus does not reduce low-field mobility relative to high-field mobility. The observed changes in the differential mobility alpha parameter exhibited by different classes of compounds when the transport gas contains a polar modifier or has a significant fraction of inert gas can be explained on the basis of the physical mechanisms involved in the separation processes.

Atmospheric pressure ion sources.

This review of atmospheric pressure ion sources discusses major developments that have occurred since 1991. Advances in the instrumentation and understanding of the key physical principles are the primary focus. Developments with electrospray and atmospheric pressure chemical ionization and variations encompassing adaptations for surface analysis, ambient air analysis, high throughput, and modification of the ionization mechanism are covered. An important and limiting consequence of atmospheric pressure chemical ionization, chemical noise, is discussed as is techniques being employed to ameliorate the problem. Ion transfer and transport from atmospheric pressure into deep vacuum is an area undergoing constant improvement and refinement so is given considerable consideration in this review.

Planar differential mobility spectrometer as a pre-filter for atmospheric pressure ionization mass spectrometry.

Ion filters based on planar DMS can be integrated with the inlet configuration of most mass spectrometers, and are able to enhance the quality of mass analysis and quantitative accuracy by reducing chemical noise, and by pre-separating ions of similar mass. This paper is the first in a series of three papers describing the optimization of DMS / MS instrumentation. In this paper the important physical parameters of a planar DMS-MS interface including analyzer geometry, analyzer coupling to a mass spectrometer, and transport gas flow control are considered. The goal is to optimize ion transmission and transport efficiency, provide optimal and adjustable resolution, and produce stable operation under conditions of high sample contamination. We discuss the principles of DMS separations and highlight the theoretical underpinnings. The main differences between planar and cylindrical geometries are presented, including a discussion of the advantages and disadvantages of RF ion focusing. In addition, we present a description of optimization of the frequency and amplitude of the DMS fields for resolution and ion transmission, and a discussion of the influence and importance of ion residence time in DMS. We have constructed a mass spectrometer interface for planar geometries that takes advantage of atmospheric pressure gas dynamic principles, rather than ion focusing, to minimize ion losses from diffusion in the analyzer and to maximize total ion transport into the mass spectrometer. A variety of experimental results has been obtained that illustrate the performance of this type of interface, including tests of resistance to high contamination levels, and the separation of stereoisomers. In a subsequent publication the control of the chemical interactions that drive the separation process of a DMS / MS system will be considered. In a third publication we describe novel electronics designed to provide the high voltages asymmetric waveform fields (SV) required for these devices as well as the effects of different waveforms.

Detection of Radiation-Exposure Biomarkers by Differential Mobility Prefiltered Mass Spectrometry (DMS-MS).

Technology to enable rapid screening for radiation exposure has been identified as an important need, and, as a part of a NIH / NIAD effort in this direction, metabolomic biomarkers for radiation exposure have been identified in a recent series of papers. To reduce the time necessary to detect and measure these biomarkers, differential mobility spectrometry - mass spectrometry (DMS-MS) systems have been developed and tested. Differential mobility ion filters preselect specific ions and also suppress chemical noise created in typical atmospheric-pressure ionization sources (ESI, MALDI, and others). Differential-mobility-based ion selection is based on the field dependence of ion mobility, which, in turn, depends on ion characteristics that include conformation, charge distribution, molecular polarizability, and other properties, and on the transport gas composition which can be modified to enhance resolution. DMS-MS is able to resolve small-molecule biomarkers from nearly-isobaric interferences, and suppresses chemical noise generated in the ion source and in the mass spectrometer, improving selectivity and quantitative accuracy. Our planar DMS design is rapid, operating in a few milliseconds, and analyzes ions before fragmentation. Depending on MS inlet conditions, DMS-selected ions can be dissociated in the MS inlet expansion, before mass analysis, providing a capability similar to MS/MS with simpler instrumentation. This report presents selected DMS-MS experimental results, including resolution of complex test mixtures of isobaric compounds, separation of charge states, separation of isobaric biomarkers (citrate and isocitrate), and separation of nearly-isobaric biomarker anions in direct analysis of a bio-fluid sample from the radiation-treated group of a mouse-model study. These uses of DMS combined with moderate resolution MS instrumentation indicate the feasibility of field-deployable instrumentation for biomarker evaluation.