Trapped Ion Mobility Mass Spectrometry Research Articles

In Vitro Inhibition of Enzymes and Antioxidant and Chemical Fingerprinting Characteristics of Azara serrata Ruiz & Pav. Fruits, an Endemic Plant of the Valdivian Forest of Chile

The World Health Organization has emphasized the importance of consuming small fruits for the prevention of chronic health problems, including diabetes, cardiovascular diseases, cancer, and obesity, which are named chronic non-communicable diseases (NCDs). Azara serrata Ruiz & Pav., commonly called “aroma de Castilla”, is a shrub endemic to Chile from the Salicaceae family that produces an underutilized blue-grey berry that grows wild in southern Chile. The species is widely used as a medicinal plant by the Andean communities of southern Chile. In this work, a high-resolution mass spectrometric analysis of the methanolic extract revealed several phenolic compounds for the first time in the edible berry of this endemic species. Furthermore, several glycosylated anthocyanins were detected and quantified using UHPLC coupled with UV/Vis detection and trapped ion mobility mass spectrometry (UHPLC-DAD-TIMS-TOF) for the anthocyanin-rich extract, which was prepared using an optimized anthocyanin extraction protocol. The extract proved to be active in the inhibition of several enzymes linked to NCDs, such as acetylcholinesterase, tyrosinase, amylase, lipase, and glucosidase (IC50 = 3.92 ± 0.23, 12.24 ± 0.03, 11.12 ± 0.10, 32.43 ± 0.0, and 371.6 ± 0.0 μg/mL, respectively). Furthermore, the extract concentrated in anthocyanins showed good antioxidant activity evidenced by the bleaching of the radicals DPPH and ABTS, ferric-reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC). The results show that these neglected endemic small berries can be a source of healthy phytochemicals. These Chilean berries can be used as functional food and their extracts are candidates for use as functional ingredients in naturally healthy products.

Label-free assessment of complement-dependent cytotoxicity of therapeutic antibodies via a whole-cell MALDI mass spectrometry bioassay

Potency assessment of monoclonal antibodies or corresponding biosimilars in cell-based assays is an essential prerequisite in biopharmaceutical research and development. However, cellular bioassays are still subject to limitations in sample throughput, speed, and often need costly reagents or labels as they are based on an indirect readout by luminescence or fluorescence. In contrast, whole-cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has emerged as a direct, fast and label-free technology for functional drug screening being able to unravel the molecular complexity of cellular response to pharmaceutical reagents. However, this approach has not yet been used for cellular testing of biologicals. In this study, we have conceived, developed and benchmarked a label-free MALDI-MS based cell bioassay workflow for the functional assessment of complement-dependent cytotoxicity (CDC) of Rituximab antibody. By computational evaluation of response profiles followed by subsequent m/z feature annotation via fragmentation analysis and trapped ion mobility MS, we identified adenosine triphosphate and glutathione as readily MS-assessable metabolite markers for CDC and demonstrate that robust concentration–response characteristics can be obtained by MALDI-TOF MS. Statistical assay performance indicators suggest that whole-cell MALDI-TOF MS could complement the toolbox for functional cellular testing of biopharmaceuticals.

Characterization of Monoclonal Antibody N-Glycan Conformations by Novel Hydrophilic Interaction Liquid Chromatography (HILIC) with Trapped Ion Mobility Mass Spectrometry (TIMS) and Fluorescence Detection (FLD)

Monoclonal antibodies (mAbs) have become prevalent in the pharmaceutical sector for treating various diseases and have a significant market presence. The determination of these molecules is complex and challenging. It is necessary to employ high-throughput technologies to characterize these pharmaceuticals in order to characterize sequencing, structure, composition, conformation, and mass. It is important to perform an N-glycosylation study on these macromolecules as their N-glycan profiles have a significant impact on their effectiveness. However, the conformational features of the N-glycans on mAb are not adequately addressed in commonly used methods. This study incorporated ion-mobility mass spectrometry as an additional dimension to established hydrophilic liquid chromatography trapped ion mobility spectrometry with fluorescence detection ((HILIC)LC/TIMS/FLD) in order to enhance the characterization of N-glycan structures. The N-glycans derived from two distinct mAbs and an immunoglobulin G (IgG) protein were labeled with procainamide and determined by (HILIC)LC/FLD with TIMS. The N-glycan profiles obtained from mAbs and IgG were examined in terms of conformation. The alterations in the N-glycan conformations were ascertained with the insertion of distinct monosaccharide units, such as galactose, into the structure. This method can be employed to clarify the intricate structures of N-glycans and offer insights into the conformational features of monoclonal antibodies throughout their production.

A Scaled Proteomic Discovery Study for Prostate Cancer Diagnostic Markers Using ProteographTM and Trapped Ion Mobility Mass Spectrometry.

There is a significant unmet need for clinical reflex tests that increase the specificity of prostate-specific antigen blood testing, the longstanding but imperfect tool for prostate cancer diagnosis. Towards this endpoint, we present the results from a discovery study that identifies new prostate-specific antigen reflex markers in a large-scale patient serum cohort using differentiating technologies for deep proteomic interrogation. We detect known prostate cancer blood markers as well as novel candidates. Through bioinformatic pathway enrichment and network analysis, we reveal associations of differentially abundant proteins with cytoskeletal, metabolic, and ribosomal activities, all of which have been previously associated with prostate cancer progression. Additionally, optimized machine learning classifier analysis reveals proteomic signatures capable of detecting the disease prior to biopsy, performing on par with an accepted clinical risk calculator benchmark.

Hyphenation of supercritical fluid chromatography and trapped ion mobility-mass spectrometry for quantitative lipidomics

BackgroundLipidomics studies require rapid separations with accurate and reliable quantification results to further elucidate the role of lipids in biological processes and their biological functions. Supercritical fluid chromatography (SFC), in particular, can provide this rapid and high-resolution separation. The combination with trapped ion mobility spectrometry (TIMS) has not yet been applied, although the post-ionization separation method in combination with liquid chromatography or imaging techniques has already proven itself in resolving isomeric and isobaric lipids and preventing false identifications. However, a multidimensional separation method should not only allow confident identification but also provide quantitative results to substantiate studies with absolute concentrations. ResultsA SFC method was developed and the hyphenation of SFC and TIMS was further explored towards the separation of different isobaric overlaps. Furthermore, lipid identification was performed using mass spectrometry (MS) and parallel accumulation serial fragmentation (PASEF) MS/MS experiments in addition to retention time and collision cross section (CCS). Quantification was further investigated with short TIMS ramps and performed based on the ion mobility signal of lipids, since TIMS increases the sensitivity by noise filtering. The final method was, as an exemplary study, applied to investigate the function of different ceramide synthases (CerS) in the nematode and model organism Caenorhabditis elegans (C. elegans). Loss of three known CerS hyl-1, hyl-2 and lagr-1 demonstrated different influences on and alterations in the sphingolipidome. SignificanceThis method describes for the first time the combination of SFC and TIMS-MS/MS, which enables a fast and sensitive quantification of lipids. The results of the application to C. elegans samples prove the functionality of the method and support research on the metabolism of sphingolipids in nematodes.

µPhos: a scalable and sensitive platform for high-dimensional phosphoproteomics

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to perturbations is crucial for further illuminating the functionality of the phosphoproteome. Here we describe µPhos (‘microPhos’), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture and small tissue amounts in <8 h total processing time. By greatly minimizing transfer steps and liquid volumes, we demonstrate increased sensitivity, >90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify ~17,000 Class I phosphosites in a human cancer cell line using 20 µg starting material, and confidently localize ~6200 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable. We employ µPhos to study drug- and time-dependent response signatures in a leukemia cell line, and by quantifying 30,000 Class I phosphosites in the mouse brain we reveal distinct spatial kinase activities in subregions of the hippocampal formation.

Ion Mobility Mass Spectrometry-Based Disaccharide Analysis of Glycosaminoglycans.

Glycosaminoglycans (GAGs) are linear and acidic polysaccharides. They are ubiquitous molecules, which are involved in a wide range of biological processes. Despite being structurally simple at first glance, with a repeating backbone of alternating hexuronic acid and hexosamine dimers, GAGs display a highly complex structure, which predominantly results from their heterogeneous sulfation patterns. The commonly applied method for compositional analysis of all GAGs is "disaccharide analysis." In this process, GAGs are enzymatically depolymerized into disaccharides, derivatized with a fluorescent label, and then analysed through liquid chromatography. The limiting factor in the high throughput analysis of GAG disaccharides is the time-consuming liquid chromatography. To address this limitation, we here utilized trapped ion mobility-mass spectrometry (TIM-MS) for the separation of isomeric GAG disaccharides, which reduces the measurement time from hours to a few minutes. A full set of disaccharides comprises twelve structures, with eight possessing isomers. Most disaccharides cannot be differentiated by TIM-MS in underivatized form. Therefore, we developed chemical modifications to reduce sample complexity and enhance differentiability. Quantification is performed using stable isotope labelled standards, which are easily available due to the nature of the performed modifications.

Four-dimensional lipidomics profiling in X-linked adrenoleukodystrophy using trapped ion mobility mass spectrometry

Lipids play pivotal roles in an extensive range of metabolic and physiological processes. In recent years, the convergence of trapped ion mobility spectrometry and MS has enabled 4D-lipidomics, a highly promising technology for comprehensive lipid analysis. 4D-lipidomics assesses lipid annotations across four distinct dimensions—retention time, collisional cross section, m/z (mass-to-charge ratio), and MS/MS spectra—providing a heightened level of confidence in lipid annotation. These advantages prove particularly valuable when investigating complex disorders involving lipid metabolism, such as adrenoleukodystrophy (ALD). ALD is characterized by the accumulation of very-long-chain fatty acids (VLCFAs) due to pathogenic variants in the ABCD1 gene. A comprehensive 4D-lipidomics strategy of ALD fibroblasts demonstrated significant elevations of various lipids from multiple classes. This indicates that the changes observed in ALD are not confined to a single lipid class and likely impacts a broad spectrum of lipid-mediated physiological processes. Our findings highlight the incorporation of mainly saturated and monounsaturated VLCFA variants into a range of lipid classes, encompassing phosphatidylcholines, triacylglycerols, and cholesterol esters. These include ultra-long-chain fatty acids with a length of up to thirty carbon atoms. Lipid species containing C26:0 and C26:1 were the most frequently detected VLCFA lipids in our study. Furthermore, we report a panel of 121 new candidate biomarkers in fibroblasts, exhibiting significant differentiation between controls and individuals with ALD. In summary, this study demonstrates the capabilities of a 4D-lipid profiling workflow in unraveling novel insights into the intricate lipid modifications associated with metabolic disorders like ALD.

Ion mobility-tandem mass spectrometry of mucin-type O-glycans

The dense O-glycosylation of mucins plays an important role in the defensive properties of the mucus hydrogel. Aberrant glycosylation is often correlated with inflammation and pathology such as COPD, cancer, and Crohn’s disease. The inherent complexity of glycans and the diversity in the O-core structure constitute fundamental challenges for the analysis of mucin-type O-glycans. Due to coexistence of multiple isomers, multidimensional workflows such as LC-MS are required. To separate the highly polar carbohydrates, porous graphitized carbon is often used as a stationary phase. However, LC-MS workflows are time-consuming and lack reproducibility. Here we present a rapid alternative for separating and identifying O-glycans released from mucins based on trapped ion mobility mass spectrometry. Compared to established LC-MS, the acquisition time is reduced from an hour to two minutes. To test the validity, the developed workflow was applied to sputum samples from cystic fibrosis patients to map O-glycosylation features associated with disease.

Abstract 7090: A scaled proteomic discovery study for prostate cancer diagnostic signatures using Proteograph workflow with trapped ion mobility mass spectrometry

Abstract Background and Aims: The low cancer specificity of serum Prostate-Specific Antigen (PSA), the principal biomarker for prostate cancer detection, leads to a high frequency of unwarranted prostate biopsies. To alleviate this deficit, we executed a proteomic discovery study seeking PSA reflex signatures using the Proteograph™ Product Suite, a multi-nanoparticle-based deep plasma/serum proteomics workflow (Seer, Inc.), with timsTOF Pro mass spectrometry (Bruker) to interrogate over 900 serum specimens from patients that underwent prostate biopsy due to elevated PSA and/or abnormal digital rectal exam. Methods: Our study followed rigorous design principles including uniform collection, randomization and blinding of specimens, all of which were processed with the Proteograph Product Suite. Liquid chromatography-mass spectrometry (LC-MS) analyses leveraged the Bruker timsTOF Pro MS platform utilizing 30-minute reversed-phase chromatography and a label-free dia-PASEF (data independent acquisition - parallel accumulation serial fragmentation) data acquisition method. Peptides deriving from a chosen subset of specimens designed to maximize peptide diversity were pooled, fractionated and analyzed using DDA (data-dependent acquisition)-PASEF to build a spectral reference library. Results: The DIA-NN algorithm was employed through the cloud-based Proteograph Analysis Suite (PAS) to search LC-MS data. Leveraging our study-specific spectral library proteomic depth achieved was approximately 3600 protein groups identified (median) per patient serum specimen and more than 4400 in at least 25% of samples across the study. Customized machine learning workflows and the SeerML pipeline were used to identify and assess diagnostic power of new proteomic signatures. Conclusions: Our data demonstrate the remarkable proteomic depth achievable in a scaled patient serum specimen discovery study using a combination of Proteograph and timsTOF platforms. This significant effort revealed serum proteomic signatures with increased diagnostic performance over that provided by PSA blood measurement and traditional patient-associated metadata alone, providing a foundation for future clinical tests aimed to reduce the current high frequency of unnecessary prostate biopsy. Citation Format: Mark Flory, Matthew Chang, Jessie May Cartier, Jane Lange, Travis Moore, James McGann, Ryan Kopp, Michael Liss, Robin Leach, Brenna Albracht, James Dai, Michael Krawitzky, Eltaher Elgierari, Jessica Chu, Paul Pease, Max Mahoney, Shadi Ferdosi, Ryan Benz, Khatereh Motamedchaboki, Asim Siddiqui, Mahdi Zamanighomi, Amir Alavi, Harendra Guturu, Daniel Hornburg, Serafim Batzoglou. A scaled proteomic discovery study for prostate cancer diagnostic signatures using Proteograph workflow with trapped ion mobility mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7090.

CE-MS/MS and CE-timsTOF to separate and characterize intramolecular disulfide bridges of monoclonal antibody subunits and their application for the assessment of subunit reduction protocols

Characterization at the subunit level enables detailed mass spectrometric characterization of posttranslational modifications (PTMs) of monoclonal antibodies (mAbs). The implemented reduction often leaves the intramolecular disulfide bridges intact. Here, we present a capillary electrophoretic (CE) method based on a neutral-coated capillary for the separation of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) digested and reduced mAb subunits followed by mass spectrometry (MS), MS/MS identification, and trapped ion mobility mass spectrometry (timsTOF). Our CE approach enables the separation of (i) different subunit moieties, (ii) various reduction states, and (iii) positional isomers of these partly reduced subunit moieties. The location of the remaining disulfide bridges can be determined by middle-down electron transfer higher energy collisional dissociation (EThcD) experiments. All these CE-separated variants show differences in ion mobility in the timsTOF measurements. Applying the presented CE-MS/MS method, reduction parameters such as the use of chaotropic salts were studied. For the investigated antibodies, urea improved the subunit reduction significantly, whereas guanidine hydrochloride (GuHCl) leads to multiple signals of the same subunit in the CE separation. The presented CE-MS method is a powerful tool for the disulfide-variant characterization of mAbs on the subunit level. It enables understanding disulfide bridge reduction processes in antibodies and potentially other proteins.

Structural characterization of steranes and hopanes in petroleum by ion mobility mass spectrometry

The structural characterization of hydrocarbon biomarkers with high molecular weight still remains a challenge even using the ultrahigh resolution mass spectrometry (UHRMS). In this work, a homologue series identification strategy was proposed based on the “sequentiality” of crude oil, where “sequentiality” revealed a compositional continuum of the naturally existing components in crude oil. With the steady increase of mass to charge ratio (m/z), the collision cross-section (CCS) value was measured by trapped ion mobility mass spectrometry (TIMS-MS) and showed a linear relationship with m/z. The structure of C20-C34 steranes and C27-C38 hopanes was determined using linear correlation. The accuracy of molecular formula was confirmed by UHRMS, and the structural determination was verified by MS/MS. The identification of sterane series and hopane series demonstrated the application of the homologue series identification strategy in the structural determination of hydrocarbon biomarkers with high molecular weight. Compared with gas chromatography-mass spectrometry (GC–MS) within the GC-able reach, the new analytical strategy showed better qualitative results and comparable quantitative results, which made it promising to explore novel hydrocarbon biomarkers in crude oil.

Tailoring Vacancy Defects in Isolated Atomically Precise Silver Clusters through Mercury-Doped Intermediates.

Vacancy defects are known to have significant effects on the physical and chemical properties of nanomaterials. However, the formation and structural dynamics of vacancy defects in atomically precise coinage metal clusters have hardly been explored due to the challenges associated with isolation of such defected clusters. Herein, we isolate [Ag28(BDT)12]2- (BDT is 1,3-benzenedithiol), a cluster with a "missing atom" site compared to [Ag29(BDT)12]3-, whose precise structure is known from X-ray diffraction. [Ag28(BDT)12]2- was formed in the gas-phase by collisional heating of [Ag28Hg(BDT)12]2-, a Hg-doped analogue of the parent cluster. The structural changes resulting from the loss of the Hg heteroatom were investigated by trapped ion mobility mass spectrometry. Density functional theory calculations were performed to provide further insights into the defect structures, and molecular dynamics simulations revealed defect site-dependent structural relaxation processes.

Ion mobility in gas and liquid phases: How much orthogonality is obtained in capillary electrophoresis-ion mobility-mass spectrometry?

Ion mobility-mass spectrometry (IM-MS) is an ever-evolving tool to separate ions in the gas phase according to electrophoretic mobility with subsequent mass determination. CE is rarely coupled to IM-MS, possibly due to similar separation mechanisms based on electrophoretic mobility. Here, we investigate the orthogonality of CE and ion mobility (IM) by analyzing a complex peptide mixture (tryptic digest of HeLa proteins) with trapped ion mobility mass spectrometry (TIMS-MS). Using the nanoCEasy interface, excellent sensitivity was achieved by identifying thousands of peptides and achieving a peak capacity of 7500 (CE: 203-323 in a 150cm long capillary, IM: 27-31). Plotting CE versus mass and CE versus (inverse) mobility, a clear grouping in curved striped patterns is observed according to the charge-to-size and mass-to-charge ratios. The peptide charge in the acidic background electrolyte can be estimated from the number of basic amino acids, with a few exceptions where neighboring effects reduce the positive charge. A surprisingly high orthogonality of CE and IM is observed, which is obviously caused by solvation effects leading to different charges and sizes in the liquid phase compared to the gas phase. A high orthogonality of CE and ion mobility is expected to be observed for other peptide samples as well as other substance classes, making CE-IM-MS a promising tool for various applications.

Sub-5-min RP-UHPLC-TIMS for high-throughput untargeted lipidomics and its application to multiple matrices.

Untargeted lipidomics, with its ability to take a snapshot of the lipidome landscape, is an important tool to highlight lipid changes in pathology or drug treatment models. One of the shortcomings of most untargeted lipidomics based on UHPLC-HRMS is the low throughput, which is not compatible with large-scale screening. In this contribution, we evaluate the application of a sub-5-min high-throughput four-dimensional trapped ion mobility mass spectrometry (HT-4D-TIMS) platform for the fast profiling of multiple complex biological matrices. Human AC-16 cells and mouse brain, liver, sclera, and feces were used as samples. By using a fast 4-min RP gradient, the implementation of TIMS allows usto differentiate coeluting isomeric and isobaric lipids, with correct precursor ion isolation, avoiding co-fragmentation and chimeric MS/MS spectra. Globally, the HT-4D-TIMS allowed usto annotate 1910 different lipid species, 1308 atthe molecular leveland 602 at thesum composition level, covering 58 lipid subclasses, together with quantitation capability covering more than three orders of magnitude. Notably, TIMS values were highly comparable with respect to longer LC gradients (CV% = 0.39%). These results highlight how HT-4D-TIMS-based untargeted lipidomics possess high coverage and accuracy, halving the analysis time with respect to conventional UHPLC methods, and can be used for fast and accurate untargeted analysis of complex matrices to rapidly evaluate changes of lipid metabolism in disease models or drug discovery campaigns.

Sliding Windows in Ion Mobility (SWIM): A New Approach to Increase the Resolving Power in Trapped Ion Mobility-Mass Spectrometry Hyphenated with Chromatography.

Over the past decade, the separation efficiency achieved by linear IMS instruments has increased substantially, with state-of-the-art IM technologies, such as the trapped ion mobility (TIMS), the cyclic traveling wave ion mobility (cTWIMS), and the structure for lossless ion manipulation (SLIM) platforms commonly demonstrating resolving powers in excess of 200. However, for complex sample analysis that require front end separation, the achievement of such high resolving power in TIMS is significantly hampered, since the ion mobility range must be broad enough to analyze all the classes of compounds of interest, whereas the IM analysis time must be short enough to cope with the time scale of the preseparation technique employed. In this paper, we introduce the concept of sliding windows in ion mobility (SWIM) for chromatography hyphenated TIMS applications that bypasses the need to use a wide and fixed IM range by using instead narrow and mobile ion mobility windows that adapt to the analytes' ion mobility during chromatographic separation. GC-TIMS-MS analysis of a mixture of 174 standards from several halogenated persistent organic pollutant (POP) classes, including chlorinated and brominated dioxins, biphenyls, and PBDEs, demonstrated that the average IM resolving power could be increased up to 40% when the SWIM mode was used, thereby greatly increasing the method selectivity for the analysis of complex samples.

Elucidating the Structures of Intermediate Fragments during Stepwise Dissociation of Monolayer-Protected Silver Clusters.

Fragmentation dynamics of ligated coinage metal clusters reflects their structural and bonding properties. So far methodological challenges limited probing structures of the fragments. Herein, we resolve the geometric structures of the primary fragments of [Ag29 L12 ]3- , i.e. [Ag24 L9 ]2- , [Ag19 L6 ]- and [Ag5 L3 ]- (L is 1,3-benzene dithiolate). For this, we used trapped ion mobility mass spectrometry to determine collision cross sections of the fragments and compared them to structures calculated by density functional theory. We also report that following two sequential [Ag5 L3 ]- elimination steps, further dissociation of [Ag19 L6 ]- also involves a new channel of Ag2 loss and Ag-S and C-S bond cleavages. This reflects a competition between retaining the electronic stability of 8 e- superatom cluster cores and increasing steric strain of ligands and staples. These results are also of potential interest for future soft-landing deposition studies aimed at probing catalytic behavior of Ag clusters on supports.

Elucidating the Structures of Intermediate Fragments during Stepwise Dissociation of Monolayer‐Protected Silver Clusters

AbstractFragmentation dynamics of ligated coinage metal clusters reflects their structural and bonding properties. So far methodological challenges limited probing structures of the fragments. Herein, we resolve the geometric structures of the primary fragments of [Ag29L12]3−, i.e. [Ag24L9]2−, [Ag19L6]− and [Ag5L3]− (L is 1,3‐benzene dithiolate). For this, we used trapped ion mobility mass spectrometry to determine collision cross sections of the fragments and compared them to structures calculated by density functional theory. We also report that following two sequential [Ag5L3]− elimination steps, further dissociation of [Ag19L6]− also involves a new channel of Ag2 loss and Ag−S and C−S bond cleavages. This reflects a competition between retaining the electronic stability of 8 e− superatom cluster cores and increasing steric strain of ligands and staples. These results are also of potential interest for future soft‐landing deposition studies aimed at probing catalytic behavior of Ag clusters on supports.

Trapped ion mobility mass spectrometry of new psychoactive substances: Isomer-specific identification of ring-substituted cathinones

New psychoactive substances (NPS) are synthetic derivatives of illicit drugs designed to mimic their psychoactive effects. NPS are typically not controlled under drug acts or their legal status depends on their molecular structure. Discriminating isomeric forms of NPS is therefore crucial for forensic laboratories. In this study, a trapped ion mobility spectrometry time-of-flight mass spectrometry (TIMS-TOFMS) approach was developed for the identification of ring-positional isomers of synthetic cathinones, a class of compounds representing two-third of all NPS seized in Europe in 2020. The optimized workflow features narrow ion-trapping regions, mobility calibration by internal reference, and a dedicated data-analysis tool, allowing for accurate relative ion-mobility assessment and high-confidence isomer identification. Ortho-, meta- and para-isomers of methylmethcathinone (MMC) and bicyclic ring isomers of methylone were assigned based on their specific ion mobilities within 5 min, including sample preparation and data analysis. The resolution of two distinct protomers per cathinone isomer added to the confidence in identification. The developed approach was successfully applied to the unambiguous assignment of MMC isomers in confiscated street samples. These findings demonstrate the potential of TIMS-TOFMS for forensic case work requiring fast and highly-confident assignment cathinone-drug isomers in confiscated samples.

Exploring Isomerism in Isolated Cyclodextrin Oligomers through Trapped Ion Mobility Mass Spectrometry.

Cyclodextrin (CD) macrocycles are used to create a wide range of supramolecular architectures which are also of interest in applications such as selective gas adsorption, drug delivery, and catalysis. However, predicting their assemblies and identifying the possible isomers in CD oligomers have always remained challenging due to their dynamic nature. Herein, we interacted CDs (α, β, and γ) with a divalent metal ion, Cu2+, to create a series of Cu2+-linked CD oligomers, from dimers to pentamers. We characterized these oligomers using electrospray ionization mass spectrometry and probed isomerism in each of these isolated oligomers using high resolution trapped ion mobility spectrometry. Using this technique, we separated multiple isomers for each of the Cu2+-interlinked CD oligomers and estimated their relative population, which was not accessible previously using other characterization techniques. We further carried out structural analysis of the observed isomers by comparing the experimental collision cross sections (CCSs) to that of modeled structures. We infer that the isomeric heterogeneity reflects size-specific packing patterns of individual CDs (e.g., close-packed/linear). In some cases, we also reveal the existence of kinetically trapped structures in the gas phase and study their transformation to thermodynamically controlled forms by examining the influence of activation of the ions on isomer interconversion.