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.

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.

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.

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.

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.

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.

Design Characteristics to Eliminate the Need for Parameter Optimization in Nanoflow ESI-MS.

The sampling efficiency in electrospray ionization-mass spectrometry (ESI-MS) can be improved by decreasing the liquid flow rate to the nanoflow regime, where it is possible to draw a large fraction of the ESI plume into the mass spectrometer. This mode of operation is typically more difficult than ESI-MS at higher flow rates because it requires careful optimization of a number of parameters to achieve optimal sampling efficiency. In this work, we screened the relative impact on signal intensity and spray stability of factors that included sprayer position, spray electrode protrusion, sprayer tip shape, spray angle relative to the MS inlet, nebulizer gas flow rate, ESI potential, and means for generating the electric field to initiate electrospray. Based on the screening results, we explore the possibility of providing fixed optimal values for many of the key source parameters to eliminate much of the tuning that is required for conventional nanoflow sources. This approach has potential to greatly simplify nanoflow ESI-MS, while providing optimized sensitivity, stability, and robustness, with decreased variability between analyses.

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.

Fast quantitation of opioid isomers in human plasma by differential mobility spectrometry/mass spectrometry via SPME/open-port probe sampling interface.

Mass spectrometry (MS) based quantitative approaches typically require a thorough sample clean-up and a decent chromatographic step in order to achieve needed figures of merit. However, in most cases, such processes are not optimal for urgent assessments and high-throughput determinations. The direct coupling of solid phase microextraction (SPME) to MS has shown great potential to shorten the total sample analysis time of complex matrices, as well as to diminish potential matrix effects and instrument contamination. In this study, we demonstrate the use of the open-port probe (OPP) as a direct and robust sampling interface to couple biocompatible-SPME (Bio-SPME) fibres to MS for the rapid quantitation of opioid isomers (i.e. codeine and hydrocodone) in human plasma. In place of chromatography, a differential mobility spectrometry (DMS) device was implemented to provide the essential selectivity required to quantify these constitutional isomers. Taking advantage of the simplified sample preparation process based on Bio-SPME and the fast separation with DMS-MS coupling via OPP, a high-throughput assay (10-15 s per sample) with limits of detection in the sub-ng/mL range was developed. Succinctly, we demonstrated that by tuning adequate ion mobility separation conditions, SPME-OPP-MS can be employed to quantify non-resolved compounds or those otherwise hindered by co-extracted isobaric interferences without further need of coupling to other separation platforms.

On the Nature of Mass Spectrometer Analyzer Contamination.

Sample throughput in electrospray ionization mass spectrometry (ESI-MS) is limited by the need for frequent ion path cleaning to remove accumulated debris that can lead to charging and general performance degradation. Contamination of ion optics within the vacuum system is particularly problematic as routine cleaning requires additional time for cycling the vacuum pumps. Differential mobility spectrometry (DMS) can select targeted ion species for transmission, thereby reducing the total number of charged particles entering the vacuum system. In this work, we characterize the nature of instrument contamination, describe efforts to improve mass spectrometer robustness by applying DMS prefiltering to reduce contamination of the vacuum ion optics, and demonstrate the capability of DMS to extend the interval between mass spectrometer cleaning. In addition, we introduce a new approach to effectively detect large charged particles formed during the electrospray ionization (ESI) process. Graphical Abstract ᅟ.

Maximizing Ion Transmission in Differential Mobility Spectrometry.

We provide modeling and experimental data describing the dominant ion-loss mechanisms for differential mobility spectrometry (DMS). Ion motion is considered from the inlet region of the mobility analyzer to the DMS exit, and losses resulting from diffusion to electrode surfaces, insufficient effective gap, ion fragmentation, and fringing field effects are considered for a commercial DMS system with 1-mm gap height. It is shown that losses due to diffusion and radial oscillations can be minimized with careful consideration of residence time, electrode spacing, gas flow rate, and waveform frequency. Fragmentation effects can be minimized by limitation of the separation field. When these parameters were optimized, fringing field effects at the DMS inlet contributed the most to signal reduction. We also describe a new DMS cell configuration that improves the gas dynamics at the mobility cell inlet. The new cell provides a gas jet that decreases the residence time for ions within the fringing field region, resulting in at least twofold increase in ion signal as determined by experimental data and simulations. Graphical Abstract ᅟ.

Correction to "Assessing Physicochemical Properties of Drug Molecules via Microsolvation Measurements with Differential Mobility Spectrometry".

[This corrects the article DOI: 10.1021/acscentsci.6b00297.].

Assessing Physicochemical Properties of Drug Molecules via Microsolvation Measurements with Differential Mobility Spectrometry.

The microsolvated state of a molecule, represented by its interactions with only a small number of solvent molecules, can play a key role in determining the observable bulk properties of the molecule. This is especially true in cases where strong local hydrogen bonding exists between the molecule and the solvent. One method that can probe the microsolvated states of charged molecules is differential mobility spectrometry (DMS), which rapidly interrogates an ion's transitions between a solvated and desolvated state in the gas phase (i.e., few solvent molecules present). However, can the results of DMS analyses of a class of molecules reveal information about the bulk physicochemical properties of those species? Our findings presented here show that DMS behaviors correlate strongly with the measured solution phase pKa and pKb values, and cell permeabilities of a set of structurally related drug molecules, even yielding high-resolution discrimination between isomeric forms of these drugs. This is due to DMS's ability to separate species based upon only subtle (yet predictable) changes in structure: the same subtle changes that can influence isomers' different bulk properties. Using 2-methylquinolin-8-ol as the core structure, we demonstrate how DMS shows promise for rapidly and sensitively probing the physicochemical properties of molecules, with particular attention paid to drug candidates at the early stage of drug development. This study serves as a foundation upon which future drug molecules of different structural classes could be examined.

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.

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.

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.