Recommendations for reporting ion mobility mass spectrometry measurements

© 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc

Correlating Resolving Power, Resolution, and Collision Cross Section: Unifying Cross-Platform Assessment of Separation Efficiency in Ion Mobility Spectrometry

Here we examine the relationship among resolving power (<i>R</i>_p), resolution (<i>R</i>_pp), and collision cross section (CCS) for compounds analyzed in previous ion mobility (IM) experiments representing a wide variety of instrument platforms and IM techniques. Our previous work indicated these three variables effectively describe and predict separation efficiency for drift tube ion mobility spectrometry experiments. In this work, we seek to determine if our previous findings are a general reflection of IM behavior that can be applied to various instrument platforms and mobility techniques. Results suggest IM distributions are well characterized by a Gaussian model and separation efficiency can be predicted on the basis of the empirical difference in the gas-phase CCS and a CCS-based resolving power definition (CCS/ΔCCS). Notably traveling wave (TWIMS) was found to operate at resolutions substantially higher than a single-peak resolving power suggested. When a CCS-based <i>R</i>_p definition was utilized, TWIMS was found to operate at a resolving power between 40 and 50, confirming the previous observations by Giles and co-workers. After the separation axis (and corresponding resolving power) is converted to cross section space, it is possible to effectively predict separation behavior for all mobility techniques evaluated (i.e., uniform field, trapped ion mobility, traveling wave, cyclic, and overtone instruments) using the equations described in this work. Finally, we are able to establish for the first time that the current state-of-the-art ion mobility separations benchmark at a CCS-based resolving power of >300 that is sufficient to differentiate analyte ions with CCS differences as small as 0.5%

Investigation of the Complete Suite of the Leucine and Isoleucine Isomers: Toward Prediction of Ion Mobility Separation Capabilities

In this study we investigated 11 isomers with the molecular formula C₆H₁₃NO₂ (<i>m</i>/<i>z</i> 131) to ascertain the potential of utilizing drift tube ion mobility mass spectrometry to aid in the separation of isomeric mixtures. This study of small molecules provides a detailed examination of the application of uniform field ion mobility for a narrow scope of isomers with variations in both bond coordination and stereochemistry. For small molecules, it was observed that in general constitutional isomers are more readily separated by uniform field mobility in comparison to stereoisomers such as enantiomers or diastereomers. Diastereomers exhibited differences in their collision cross section (CCS), but were unresolvable in a mixture, whereas the enantiomers studied did not exhibit statistically different CCS values. A mathematical relationship relating the CCS to resolving power was developed in order to predict the required ion mobility resolving power needed to separate the various isomer classes. For the majority of isomers evaluated in this study, a uniform field-based resolving power of 100 was predicted to be sufficient to resolve over half (∼60%) of all hypothetical isomer pairs, including leucine and isoleucine, whereas their stereoisomers (d- and l-forms) are predicted to be significantly more challenging, if not impossible, to separate by conventional drift tube techniques

Evaluation of Collision Cross Section Calibrants for Structural Analysis of Lipids by Traveling Wave Ion Mobility-Mass Spectrometry

Collision cross section (CCS) measurement of lipids using traveling wave ion mobility-mass spectrometry (TWIM-MS) is of high interest to the lipidomics field. However, currently available calibrants for CCS measurement using TWIM are predominantly peptides that display quite different physical properties and gas-phase conformations from lipids, which could lead to large CCS calibration errors for lipids. Here we report the direct CCS measurement of a series of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in nitrogen using a drift tube ion mobility (DTIM) instrument and an evaluation of the accuracy and reproducibility of PCs and PEs as CCS calibrants for phospholipids against different classes of calibrants, including polyalanine (PolyAla), tetraalkylammonium salts (TAA), and hexakis­(fluoroalkoxy)­phosphazines (HFAP), in both positive and negative modes in TWIM-MS analysis. We demonstrate that structurally mismatched calibrants lead to larger errors in calibrated CCS values while the structurally matched calibrants, PCs and PEs, gave highly accurate and reproducible CCS values at different traveling wave parameters. Using the lipid calibrants, the majority of the CCS values of several classes of phospholipids measured by TWIM are within 2% error of the CCS values measured by DTIM. The development of phospholipid CCS calibrants will enable high-accuracy structural studies of lipids and add an additional level of validation in the assignment of identifications in untargeted lipidomics experiments

Solvent Composition Can Have a Measurable Influence on the Ion Mobility-Derived Collision Cross Section of Small Molecules

Ion mobility (IM) is an important analytical technique for increasing identification coverage of metabolites in untargeted studies, especially when integrated into traditional liquid chromatography–mass spectrometry workflows. While there has been extensive work surrounding best practices to obtain and standardize collision cross section (CCS) measurements necessary for comparing across different IM techniques and laboratories, there has been little investigation into experimental factors beyond the mobility separation region that could potentially influence CCS measurements. The first-principles derived CCS of 15 chemical standards were evaluated across 27 aqueous:organic solvent compositions using a high-precision drift tube instrument. A small but measurable dependency of the CCS on the solvent composition was observed, with the larger analytes from this study (m/z > 400) exhibiting a characteristic increase in CCS at the intermediate (40–60%) solvent compositions. Parallels to the behavior of solvent viscosity and protonation site tautomers (protomers) were noted, although the origin of these solvent-dependent CCS trends is as yet unclear. Taken together, these findings document a solvent dependency on CCS, which, while minor (<0.5%), identifies an important need for reporting the solvent system when utilizing CCS in comparative ion mobility studies

Determining Double Bond Position in Lipids Using Online Ozonolysis Coupled to Liquid Chromatography and Ion Mobility-Mass Spectrometry

The increasing focus on lipid metabolism has revealed a need for analytical techniques capable of structurally characterizing lipids with a high degree of specificity. Lipids can exist as any one of a large number of double bond positional isomers, which are indistinguishable by single-stage mass spectrometry alone. Ozonolysis reactions coupled to mass spectrometry have previously been demonstrated as a means for localizing double bonds in unsaturated lipids. Here we describe an online, solution-phase reactor using ozone produced via a low-pressure mercury lamp, which generates aldehyde products diagnostic of cleavage at a particular double bond position. This flow-cell device is utilized in conjunction with structurally selective ion mobility-mass spectrometry. The lamp-mediated reaction was found to be effective for multiple lipid species in both positive and negative ionization modes, and the conversion efficiency from precursor to product ions was tunable across a wide range (20–95%) by varying the flow rate through the ozonolysis device. Ion mobility separation of the ozonolysis products generated additional structural information and revealed the presence of saturated species in a complex mixture. The method presented here is simple, robust, and readily coupled to existing instrument platforms with minimal modifications necessary. For these reasons, application to standard lipidomic workflows is possible and aids in more comprehensive structural characterization of a myriad of lipid species

Determining Double Bond Position in Lipids Using Online Ozonolysis Coupled to Liquid Chromatography and Ion Mobility-Mass Spectrometry

The increasing focus on lipid metabolism has revealed a need for analytical techniques capable of structurally characterizing lipids with a high degree of specificity. Lipids can exist as any one of a large number of double bond positional isomers, which are indistinguishable by single-stage mass spectrometry alone. Ozonolysis reactions coupled to mass spectrometry have previously been demonstrated as a means for localizing double bonds in unsaturated lipids. Here we describe an online, solution-phase reactor using ozone produced via a low-pressure mercury lamp, which generates aldehyde products diagnostic of cleavage at a particular double bond position. This flow-cell device is utilized in conjunction with structurally selective ion mobility-mass spectrometry. The lamp-mediated reaction was found to be effective for multiple lipid species in both positive and negative ionization modes, and the conversion efficiency from precursor to product ions was tunable across a wide range (20–95%) by varying the flow rate through the ozonolysis device. Ion mobility separation of the ozonolysis products generated additional structural information and revealed the presence of saturated species in a complex mixture. The method presented here is simple, robust, and readily coupled to existing instrument platforms with minimal modifications necessary. For these reasons, application to standard lipidomic workflows is possible and aids in more comprehensive structural characterization of a myriad of lipid species

Structural Characterization of Methylenedianiline Regioisomers by Ion Mobility-Mass Spectrometry, Tandem Mass Spectrometry, and Computational Strategies. 3. MALDI Spectra of 2-Ring Isomers

Characterization of methylenedianiline (MDA) 2-ring isomers (2,2′-, 2,4′-, and 4,4′-MDA) is reported using matrix assisted laser desorption/ionization–mass spectrometry (MALDI-MS), a common technique used for characterizing synthetic polymers. MDA is a precursor to methylene diphenyl diisocyanate (MDI), a hard block component in polyurethane (PUR) synthesis. This work focuses on comparing MALDI results to those of our previous electrospray ionization-mass spectrometry (ESI-MS) studies. In ESI, 2-ring MDA isomers formed single unique [M + H]⁺ (199 Da) parent ions, whereas in MALDI each isomer shows significant formation of three precursor ions: [M – H]⁺ = 197 Da, [M^•]⁺ = 198 Da, and [M + H]⁺ = 199 Da. Structures and schemes are proposed for the MALDI fragment ions associated with each precursor ion. Ion mobility–mass spectrometry (IM-MS), tandem mass spectrometry (MS/MS), and computational methods were all critical in determining the structures for both precursor and fragment ions as well as the fragmentation mechanisms. The present study indicates that the [M – H]⁺ and [M^•]⁺ ions are formed by the MALDI process, explaining why they were not observed with ESI

Structural Characterization of Methylenedianiline Regioisomers by Ion Mobility-Mass Spectrometry, Tandem Mass Spectrometry, and Computational Strategies: I. Electrospray Spectra of 2‑Ring Isomers

Purified methylenedianiline (MDA) regioisomers were structurally characterized and differentiated using tandem mass spectrometry (MS/MS), ion mobility-mass spectrometry (IM-MS), and IM-MS/MS in conjunction with computational methods. It was determined that protonation sites on the isomers can vary depending on the position of amino groups, and the resulting protonation sites play a role in the gas-phase stability of the isomer. We also observed differences in the relative distributions of protonated conformations depending on experimental conditions and instrumentation, which is consistent with previous studies on aniline in the gas phase. This work demonstrates the utility of a multifaceted approach for the study of isobaric species and elucidates why previous MDA studies may have been unable to detect and/or differentiate certain isomers. Such analysis may prove useful in the characterization of larger MDA multimeric species, industrial MDA mixtures, and methylene diphenyl diisocyanate (MDI) mixtures used in polyurethane synthesis

Structural Characterization of Methylenedianiline Regioisomers by Ion Mobility-Mass Spectrometry, Tandem Mass Spectrometry, and Computational Strategies. 2. Electrospray Spectra of 3‑Ring and 4‑Ring Isomers

Building on results from our previous study of 2-ring methylenedianiline (MDA), a combined mass spectrometry approach utilizing ion mobility-mass spectrometry (IM-MS) and tandem mass spectrometry (MS/MS) coupled with computational methods enables the structural characterization of purified 3-ring and 4-ring MDA regioisomers in this current study. The preferred site of protonation for the 3-ring and 4-ring MDA was determined to be on the amino groups. Additionally, the location of the protonated amine along the MDA multimer was found to influence the gas phase stability of these molecules. Fragmentation mechanisms similar to the 2-ring MDA species were observed for both the 3-ring and 4-ring MDA. The structural characterization of 3-ring and 4-ring MDA isomers using modern MS techniques may aid polyurethane synthesis by the characterization of industrial grade MDA, multimeric MDA species, and methylene diphenyl diisocyanate (MDI) mixtures