The Role of IM-HRMS Outside Proteomics

While proteomics researchers hunt for peptides representing predetermined protein group identifications (IDs), the non-proteomics community wrestles with the “dark omics” more directly: a chaotic landscape of uncharacterized small molecules, vast dynamic ranges, and structural isomers that defy identification by liquid-chromatography-high-resolution mass spectrometry (LC–HRMS) alone. Instead of focusing on what is being missed, time is spent determining whether what is being measured is accurate, precise, and correctly identified. More compounds? Not necessarily. More confidence? Absolutely.

By looking beyond the proteomics-driven ultra-high-end tier of instrumentation, instrument manufacturers can bring substantial value by democratizing novel separation solutions, including ion-mobility-mass spectrometry (IM-MS). When applied with intention to a growing list of high-resolution mass spectrometry (HRMS) applications, IM-MS and high-resolution versions represent the potential to directly address unmet needs in separation, speed, sensitivity, and confident identification across a much larger and underserved analytical market. To move this discussion from principle to practice, it is helpful to examine concrete examples, where ion mobility mass spectrometry with high-resolution mass spectrometry (IM-HRMS) has already shifted analytical outcomes. Rather than speaking abstractly about separation power and confidence, the following case studies illustrate how IM-HRMS directly resolves real bottlenecks in regulated and high-stakes environments. These examples demonstrate that the value of IM-MS is no longer theoretical but is already reshaping workflows in pharmaceutical development and toxicology, where confident compound discrimination and identification are essential.

Pharmaceutical Drug Development

The characterization of complex stereoisomers, such as epimers in antibody-drug conjugates (ADCs) and atropisomers in targeted cancer therapeutics, represents a critical bottleneck in modern pharmaceutical development. Stereochemistry fundamentally dictates biological activity and drug safety; for example, the precise configuration of a citrulline-based ADC linker governs its enzymatic cleavage for targeted payload release¹ while the atropisomeric purity of covalent inhibitors like GDC-6036 is an essential quality attribute.² Failing to accurately resolve and quantify these undesired isomers compromises synthetic route optimization and therapeutic efficacy.

Historically, distinguishing these isomeric molecules in a routine workflow was immensely challenging. Reverse-phase liquid chromatography (RP-LC) often leads to coelution of isomeric mixtures, and tandem mass spectrometry (MS/MS) cannot differentiate them because of identical precursor masses and chimeric fragmentation patterns. While chiral chromatography can sometimes force a separation, it demands exhaustive, resource-intensive method development, suffers from long run times, and utilizes mobile phases that are frequently incompatible with mass spectrometry. Furthermore, traditional lower-resolution ion mobility platforms lack the resolving power necessary to separate these closely related species in complex mixtures.

The authors overcame these hurdles with IM-HRMS by separating ions based on subtle differences in their three-dimensional gas-phase size, shape, and charge, a property known as the collision cross section (CCS). By utilizing extended effective path lengths, IM-HRMS achieved exceptional resolving power and successfully achieved baseline resolution of the previously referenced coeluting ADC linker epimers and GDC-6036 atropisomers. The seamless integration of IM-HRMS into standard LC–MS workflows can provide a robust, single-run analytical solution that bypasses traditional limitations. This approach eliminates reliance on intensive chiral chromatography screening, enabling rapid, high-throughput structural elucidation and direct relative quantitation, which can significantly accelerate quality control and advanced biopharmaceutical manufacturing.

Toxicology

The clinical toxicology landscape is increasingly complicated by polysubstance use and the rapid emergence of novel psychoactive substances (NPSs).³ While LC–HRMS provides powerful untargeted screening capabilities to detect these unpredictable exposures, it faces a critical limitation: the inability to easily differentiate structural isomers and matrix interferences. A prominent real-world consequence is the clinical and forensic analysis of fentanyl analogs. Laboratories routinely struggle to quickly and reliably differentiate positional isomers such as ortho-, meta-, and para-fluorofentanyl, or structurally similar compounds like cyclopropyl and crotonyl fentanyl.

Differentiating these illicit analogs is vital because stereochemical and structural variations dictate vastly different pharmacological potencies and toxicities. Failing to accurately identify the exact isomer potentially limits the ability to determine appropriate medical responses, obscures critical epidemiological data regarding the illicit drug supply, and hinders accurate forensic and legal proceedings. Researchers used IM-HRMS to directly address this isomeric challenge by separating gas-phase ions based on their CCS as highlighted previously. Furthermore, the authors have discovered that by deliberately forming nonconventional metal cation adducts (such as sodium, lithium, or copper) during ionization, IM-HRMS amplifies the conformational differences between isomers, achieving baseline separation of species that perfectly coelute in standard LC–MS.⁴

The potential integration of IM-HRMS into clinical, forensic, and public health screening workflows offers a profound leap forward for toxicology. It bypasses the need for exhaustively long and complex chromatographic methods while delivering highly confident, rapid identification and quantification of drugs and metabolites. Ultimately, IM-HRMS can empower laboratories to perform truly comprehensive surveillance, keeping public health responses agile against a rapidly mutating drug supply.

Highlighting the Need for Divergence

While these examples showcase clear technical victories, they also expose a broader structural issue in the market. Laboratories that would benefit from these capabilities are often anchored to legacy workflows optimized around triple quadrupole mass spectrometry (QqQ-MS) platforms and existing HRMS platforms. As evidence mounts, the question is no longer whether IM-MS works, but rather why does adoption remain uneven across non-proteomics domains? Understanding this divergence requires an appreciation of the practical, regulatory, and economic barriers that define decision-making in these laboratories.

Non-proteomics customers face a unique set of barriers that prevent the transition from the gold standard of QqQ-MS analyses to the information-rich world of IM-enabled HRMS. Unlike the typical proteomics customer who may never envision a QqQ or standard HRMS system meeting their needs, it is not uncommon for an internal debate to cyclically resurface in organizations that are dominated by legacy technology yet yearning for the value that IM-enabled instruments provide. In many regulated settings, IM-MS-enabled HRMS is unlikely to displace triple quadrupole instruments for high-volume routine quantitation. Rather, it may be complementary in serving as a confirmatory, characterization, and method-development catalyst that reduces ambiguity and improves confidence in complex or evolving analyte panels.

The Reality of the Price and Performance Trade-Off

Any discussion of expanded IM-MS adoption must first confront the economic reality facing most non-proteomics laboratories. IM-MS-enabled HRMS platforms carry higher upfront capital costs, service contracts, and training requirements compared to workflows that are already validated and deeply embedded in existing environments. For many laboratories, the question is not whether IM-HRMS performs better in select applications, but whether the gains in isomer discrimination, interference reduction, and improved data quality translate into measurable operational or financial return. In some cases, replacing lengthy chiral separations, reducing confirmatory testing, shortening method development cycles, or preventing costly misidentifications can provide justification for the investment. In others, legacy workflows may remain economically sufficient despite their known limitations. For laboratories that have not yet fully recognized how IM-MS can enable faster decisions, reduce downstream costs, and strengthen long-term competitiveness, the practical challenges outlined in this article illustrate where performance advantages can provide clear economic justification.

The Sensitivity and Specificity Gap

For many environments, including food safety and clinical toxicology, sensitivity and specificity are crucial. HRMS instruments have historically struggled to match the sensitivity and linear dynamic range of QqQ platforms operating in selected reaction monitoring (SRM) mode. However, IM-HRMS offers a solution not only by increasing ion utilization, but by cleaning the window through which we view the sample.

By separating ions based on size, shape, and charge in the gas phase, IM-HRMS offers an efficient separation and time compression, removing chemical noise and matrix interferences that plague complex samples like urine or food extracts. For example, in environmental analysis, the coupling of IM-MS to HRMS has been shown to reduce background noise significantly, enhancing the signal-to-noise ratio and potentially allowing HRMS to compete with the sensitivity of targeted methods while retaining the benefits of non-targeted screening.⁵

The Isomer Challenge: When Mass Resolution Hits a Wall

Perhaps the most critical argument for the widespread adoption of IM-HRMS is the “Isomer Challenge." Despite resolving powers exceeding 100,000 FWHM, mass spectrometry alone cannot distinguish distinct isomers. This is a pervasive issue across all domains:

Clinical & Toxicology Applications

Vitamin D deficiency testing is hampered by isomeric metabolites. Recent work using technology capable of mobility resolving powers greater than 200 separated specific vitamin D metabolite conformers and isomers that were previously obscured in low-resolution IM-MS, offering a path toward interference-free quantitation. Similarly, in toxicology, separating fentanyl isomers and analogs is critical for legal and medical outcomes; IM-HRMS has demonstrated the ability to separate these isomers, particularly when aided by cation-adduct strategies.⁶

Metabolomics/Lipidomics

In metabolomics, researchers analyze complex biological mixtures plagued by the pervasive presence of structural and stereoisomers, including lipid, amino acid, bile acid, and vitamin isomers.⁷ Often, these isomers perfectly coelute in standard LC and yield indistinguishable tandem mass spectra. In lipidomics, the biological function of lipids is dictated by double-bond positions and sn-positional isomers, an area where standard LC–MS often fails. Interlaboratory studies using structures for lossless ion manipulation (SLIM)-based IM-HRMS have demonstrated the ability to separate challenging lipid isomers, such as triglycerides, which exhibit multiple mobility features that LC cannot resolve.⁸

Oligonucleotide Therapeutics

As drug development shifts toward large molecules, characterizing phosphorothioate (PS) oligonucleotides has become increasingly complex due to diastereomer generation. Recent IM-HRMS applications have achieved baseline separation of these diastereomers, a feat impossible with standard MS, ensuring better quality control for these complex therapeutics.⁹

Environmental (PFAS)

The analysis of per- and polyfluoroalkyl substances (PFAS) requires distinguishing between linear and branched isomers to understand toxicity and environmental fate. IM-HRMS has proven capable of separating these isomers where traditional LC–MS struggles, providing a clearer picture of environmental contamination.¹⁰

Exposomics

The rapidly emerging field of exposomics aims to map the vast array of environmental chemicals influencing human health, but is similarly hindered by unannotated features and the inability to resolve complex structural isomers. As exposomics transitions from proof-of-concept to large-scale population studies, IM-HRMS technology can be a critical accelerator. Ultimately, IM-HRMS has shown potential for researchers to comprehensively illuminate missing features and enable rapid, isomer-specific environmental surveillance to accelerate our understanding of the non-genetic drivers of chronic disease.^11,12

The Illuminating Sides of IM-HRMS and CCS

A major bottleneck in metabolomics and exposomics is the number of spectral features that remain unannotated due to gaps in spectral libraries. A similar bottleneck exists for drug metabolism studies that require structural confirmation of new drug metabolites across multiple studies and animal species, including humans. Retention time (tR) is notoriously variable between labs, across instruments in the same laboratory, and even within different lots of the same column, making database sharing of tR difficult. CCS represents a more robust physicochemical property that is largely independent of instrumentation and mobile phase conditions, with full harmonization an active area of research.¹³ Including CCS in libraries adds a high-confidence identification parameter that can filter out false positives. In IM-MS, just as in liquid chromatography, peak capacity defines the maximum number of resolvable peaks that can fit within a separation window. To successfully characterize complex mixtures, achieving high resolving power simultaneously across the entire mass and mobility range is the absolute key and one of the differentiators of the SLIM technology.¹⁴

Non-Proteomics Laboratories

The ideal state for a non-proteomics laboratory is not necessarily an instrument with all the bells and whistles, but is likely an instrument that provides:

• Simplified Workflows: Replacing complex sample prep with gas-phase cleaning via IM-MS to handle "dirty" matrices in food and environmental samples.
• Better and More Accessible Data: Integrating 4D data (tR, m/z, Intensity, CCS) seamlessly into vendor and vendor-neutral software to lower the barrier to entry for harnessing higher quality data.
• Unambiguous Identifications: Resolving isomers (drug metabolites, PFAS, lipids) that currently require hour-long LC gradients or are simply ignored.

Importantly, it is critical to recognize that "high end" does not have to mean "complex" or "proteomics-led." Technologies like iterative SLIM (itSLIM) are proving that we can achieve ultra-high resolution separations (effective path lengths of >100 meters) on compact footprints, enabling the separation of even the most difficult small and large compounds without extending physical bench space.¹⁵ Thankfully, the technology to solve the problems included here exists. It is time to design it for the analysts identifying pesticides in our food, drugs in our system, pollutants in our water, and the totality of exposures in chronic disease. Revealing what others leave unseen with greater speed, accuracy, precision, and confidence is likely where the next revolution lies.

References

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2. Varona, M.; Dobson, D. P.; Napolitano, J. G. et al. High Resolution Ion Mobility Enables the Structural Characterization of Atropisomers of GDC-6036, a KRAS G12C Covalent Inhibitor. J. Am. Soc. Mass Spectrom. 2024. 35 (11), 2586-2595.DOI: 10.1021/jasms.4c00103
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