High-resolution Accurate Mass Measurements Research Articles

Novel anthraquinone α-aryl-α-aminophosphonates: Synthesis, spectroscopy and imaging by confocal laser scanning microscopy of trematode Opisthioglyphe ranae

A small library of new anthraquinone α-aryl-α-aminophosphonates with N-C-P molecular fragment was synthesized under Kabachnik-Fields one-pot solvent-free reaction conditions from 1-aminoanthraquinone. 1H-, 13C-, 31P NMR and FTIR spectroscopy and high-resolution accurate mass measurement was employed to confirm structures of new dyes. The photophysical parameters of the studied α-aminophosphonates have been investigated by means of UV–Vis and fluorescence spectroscopy in organic solvents of varying polarity. Obtained compounds were found to emit light from 586 nm (EtOAc) to 620 nm (DMSO) with fluorescence bathochromic shifts reaching up to 30 nm, Stokes shift of 4573 cm– 1 and photostability of up to 60 % of initial intensity upon photofading for 4 h. α-Aryl-α-aminophosphonate was used for the first time as a dye for rapid bioimaging of trematode Opisthioglyphe ranae in confocal laser scanning microscopy.

Separation of Isobaric Lipids in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions.

The diverse array of chemical compounds present in tissue samples results in many isobaric (i.e., same nominal mass) compounds in imaging mass spectrometry experiments. Adequate separation and differentiation of these compounds is necessary to ensure accurate analyte identification and avoid composite images comprising multiple compounds. High-resolution accurate mass (HRAM) measurements are able to resolve these compounds in some instances, but HRAM measurements are not always feasible depending on the instrument platform and the desired experimental time scale. Alternatively, tandem mass spectrometry (MS/MS) can be used to perform gas-phase transformations that improve molecular specificity. While conventional MS/MS methods employ collision induced dissociation (CID) to fragment compounds of interest and then analyze the product masses, gas-phase ion/ion reactions can be used to instead selectively react with desired classes of analytes. Herein, we have used gas-phase charge inversion ion/ion reactions to selectively resolve phosphatidylcholines (PCs) in isobaric lipid mixtures. A 1,4-phenylenedipropionic acid (PDPA) reagent dianion readily reacts with [M + H]+, [M + Na]+, and [M + K]+ ion types to produce demethylated product anions for each PC, [PC - CH3]-. These product anions are no longer isobaric and now differ in mass by 22 Da (protonated versus sodiated) and 16 Da (sodiated versus potassiated), respectively. This reaction has been used to differentiate isobaric lipids in the imaging mass spectrometry analysis of rat brain tissue.

193 nm Ultraviolet Photodissociation for the Characterization of Singly Charged Proteoforms Generated by MALDI.

MALDI imaging allows for the near-cellular profiling of proteoforms directly from microbial, plant, and mammalian samples. Despite detecting hundreds of proteoforms, identification of unknowns with only intact mass information remains a distinct challenge, even with high mass resolving power and mass accuracy. To this end, many supplementary methods have been used to create experimental databases for accurate mass matching, including bulk or spatially resolved bottom-up and/or top-down proteomics. Herein, we describe the application of 193 nm ultraviolet photodissociation (UVPD) for fragmentation of quadrupole isolated singly charged ubiquitin (m/z 8565) by MALDI-UVPD on a UHMR HF Orbitrap. This platform permitted the high-resolution accurate mass measurement of not just terminal fragments but also large internal fragments. The outlined workflow demonstrates the feasibility of top-down analyses of isolated MALDI protein ions and the potential toward more comprehensive characterization of proteoforms in MALDI imaging applications.

Enhanced Spatial Mapping of Histone Proteoforms in Human Kidney Through MALDI-MSI by High-Field UHMR-Orbitrap Detection.

Core histones including H2A, H2B, H3, and H4 are key modulators of cellular repair, transcription, and replication within eukaryotic cells, playing vital roles in the pathogenesis of disease and cellular responses to environmental stimuli. Traditional mass spectrometry (MS)-based bottom-up and top-down proteomics allows for the comprehensive identification of proteins and of post-translational modification (PTM) harboring proteoforms. However, these methodologies have difficulties preserving near-cellular spatial distributions because they typically require laser capture microdissection (LCM) and advanced sample preparation techniques. Herein, we coupled a matrix-assisted laser desorption/ionization (MALDI) source with a Thermo Scientific Q Exactive HF Orbitrap MS upgraded with ultrahigh mass range (UHMR) boards for the first demonstration of complementary high-resolution accurate mass (HR/AM) measurements of proteoforms up to 16.5 kDa directly from tissues using this benchtop mass spectrometer. The platform achieved isotopic resolution throughout the detected mass range, providing confident assignments of proteoforms with low ppm mass error and a considerable increase in duty cycle over other Fourier transform mass analyzers. Proteoform mapping of core histones was demonstrated on sections of human kidney at near-cellular spatial resolution, with several key distributions of histone and other proteoforms noted within both healthy biopsy and a section from a renal cell carcinoma (RCC) containing nephrectomy. The use of MALDI-MS imaging (MSI) for proteoform mapping demonstrates several steps toward high-throughput accurate identification of proteoforms and provides a new tool for mapping biomolecule distributions throughout tissue sections in extended mass ranges.

Pharmaceuticals are identified in insects in River Fyris – A study with both tandem quadrupole and quadrupole-time-of-flight mass spectrometry

The internal concentrations of active pharmaceutical compounds in insects/macroinvertebrates in River Fyris, Uppsala, Sweden, have for the first time been investigated. Specimen of backswimmer, caddisfly larva, damselfly larva, mayfly larva and water louse were caught in the river downstream a waste-water treatment plant. After homogenization and extraction, analysis was carried out with Ultra-High Performance Liquid Chromatography – Electrospray – Tandem Mass Spectrometry (UHPLC-ESI-MS/MS) using both a tandem quadrupole (QqQ) and a quadrupole-time-of-flight (qToF) mass spectrometer. A combined qualitative/quantitative screening method for 89 pharmaceutical compounds in the selective reaction monitoring mode was applied to the samples using the QqQ instrument. Thirty-three different drugs were detected and quantified in the macroinvertebrate samples, covering a wide range of pharmacological classes, the most common being antidepressants, psycholeptics and antihistamines. Drug concentrations up to 260 ng/g (for tetracycline) were determined. A subset of thirteen compounds were selected for a complementary qualitative UHPLC-qToF-MS/MS analysis. This way, the confidence in the substance identifications was significantly strengthened by the availability of full scan high-resolution spectra and accurate mass measurements. This is the first study demonstrating the uptake of pharmaceuticals in water-living macroinvertebrates in Sweden. Since these organisms are in the lower part of the food chain, pharmaceuticals might accumulate in predators leading to ecotoxicological effect at higher trophic levels. Macroinvertebrates might be promising indicator organisms for pharmaceutical pollution.

Comparison of High-Resolution Fourier Transform Mass Spectrometry Platforms for Putative Metabolite Annotation.

Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap mass spectrometry (MS) are among the highest-performing analytical platforms used in metabolomics. Non-targeted metabolomics experiments, however, yield extremely complex datasets that make metabolite annotation very challenging and sometimes impossible. The high-resolution accurate mass measurements of the leading MS platforms greatly facilitate this process by reducing mass errors and spectral overlaps. When high resolution is combined with relative isotopic abundance (RIA) measurements, heuristic rules, and constraints during searches, the number of candidate elemental formula(s) can be significantly reduced. Here, we evaluate the performance of Orbitrap ID-X and 12T solariX FT-ICR mass spectrometers in terms of mass accuracy and RIA measurements and how these factors affect the assignment of the correct elemental formulas in the metabolite annotation pipeline. Quality of the mass measurements was evaluated under various experimental conditions (resolution: 120, 240, 500 K; automatic gain control: 5 × 104, 1 × 105, 5 × 105) for the Orbitrap MS platform. High average mass accuracy (<1 ppm for UPLC-Orbitrap MS and <0.2 ppm for direct infusion FT-ICR MS) was achieved and allowed the assignment of correct elemental formulas for over 90% (m/z 75-466) of the 104 investigated metabolites. 13C1 and 18O1 RIA measurements further improved annotation certainty by reducing the number of candidates. Overall, our study provides a systematic evaluation for two leading Fourier transform (FT)-based MS platforms utilized in metabolite annotation and provides the basis for applying these, individually or in combination, to metabolomics studies of biological systems.

Molecular-Formula Determination through Accurate-Mass Analysis: A Forensic Investigation

Mass spectrometry is frequently introduced to undergraduate students as an instrument for both qualitative and quantitative chemical analysis. One of the most common uses of mass spectrometry (MS) is to deduce or confirm a compound’s chemical formula, by relying on high-resolution accurate-mass measurements. However, like all forms of instrumental analysis, qualitative characterization of an unknown entails a degree of measurement uncertainty. By extension, the conclusions of any analysis may not be definitive. Here, we describe a form of inquiry-based learning wherein students acquire scientific evidence to probe a question (“Is the person guilty?”). A laboratory-based experiment involves the use of accurate-mass analysis to characterize a trace unknown, extracted from “simulated” forensic evidence. Together with the accurate mass, the isotopic distribution, some chemical intuition, and an online molecular-formula tool (available through ChemCalc), students arrive at the suspected chemical identity of the unknown. The analysis of cocaine on circulating currency is used in this report to illustrate the lab exercise. At the conclusion of the exercise, students should realize that the evidence they generate is insufficient to support a definitive identification. The strategies employed to deduce a molecular formula may also be adopted to a classroom setting.

Sequencing Proteins from Bottom to Top: Combining Techniques for Full Sequence Analysis of Glucokinase.

Proteomics-based mass spectrometry has gained increasing amounts of popularity in recent years. In particular, high resolution accurate mass measurements in mass spectrometry has gained notoriety for giving the capability of high throughput analysis with lower cost to the user. In particular, its uses in the identification of protein sequence through the utilization of bottom-up, middle-down, and top-down approaches has been widely discussed. In this chapter, we discuss the advantages of each technique as well as using the techniques in tandem to gain well-rounded structural data on our protein of interest, glucokinase. The study will focus on the use of Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry, but give insights into the advantages that may come from the utilization of other high resolution techniques.

Multiple TOF/TOF events in a single laser shot for multiplexed lipid identifications in MALDI imaging mass spectrometry

Tandem mass spectrometry (MS/MS) is often used to identify lipids in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) workflows. The molecular specificity afforded by MS/MS is crucial on MALDI time-of-flight (TOF) platforms that generally lack high resolution accurate mass measurement capabilities. Unfortunately, imaging MS/MS workflows generally only monitor a single precursor ion over the imaged area, limiting the throughput of this methodology. Herein, we demonstrate that multiple TOF/TOF events performed in each laser shot can be used to improve the throughput of imaging MS/MS. This is shown to enable the simultaneous identification of multiple phosphatidylcholine lipids in rat brain tissue. Uniquely, the separation in time achieved for the precursor ions in the TOF-1 region of the instrument is maintained for the fragment ions as they are analyzed in TOF-2, allowing for the differentiation of fragment ions of the exact same m/z derived from different precursor ions (e.g., the m/z 163 fragment ion from precursor ion m/z 772.5 is easily distinguished from the m/z 163 fragment ion from precursor ion m/z 826.5). This multiplexed imaging MS/MS approach allows for the acquisition of complete fragment ion spectra for multiple precursor ions per laser shot.

Fragmentation behavior of DOTA complexes under different activation conditions.

We have investigated the fragmentation behavior of a number of DOTA-metal complexes under collision-induced dissociation, infrared-multiphoton dissociation and higher-energy collisional dissociation activation conditions. Both, positive and negative ion mode electrospray ionization was applied. The results show that characteristic fragmentations were obtained for the metal-complexes under the investigated conditions. All elemental compositions of fragment ions have been unambiguously identified by high resolution-accurate mass measurements. Certain trends, for instance eliminations of carbon dioxide, alkyl and amine residues, were observed that coincide with the size of the metal and its location within the periodic table. Additionally, lanthanide, aluminium and indium species with even oxidation state or containing a radical have been detected. To further investigate the observed water capture during activation, deuterium labeling experiments have also been carried out. Copyright © 2017 John Wiley & Sons, Ltd.

Identification of porcine relaxin in plasma by liquid chromatography-high resolution mass spectrometry.

Relaxin (RLX) has demonstrated diverse pharmacological effects in various scientific and clinical studies. The emergence of porcine relaxin (pRLX) has raised concerns on the doping potential of pRLX. There have also been speculations in the horseracing industry on its covert use. To control the abuse of pRLX in equine sports, a method to detect pRLX effectively and to provide its unequivocal identification in equine biological samples is required. This paper reports on the detection and confirmation of pRLX in equine plasma by liquid chromatography-high resolution mass spectrometry. pRLX was isolated from equine plasma by immunoaffinity purification using anti-pRLX antibody-coated magnetic beads. Anti-pRLX antibody was generated in-house by purifying antisera from rabbits immunized with pRLX. The isolated pRLX was subjected to reduction of their disulfide bonds to obtain their respective A- and B-chains. The extracts were then further purified and concentrated prior to reversed-phase LC separation and high resolution accurate mass measurement. As detection of the A-chains was far more sensitive than that of the B-chains, the A-chain of pRLX was set as the targets for detection and confirmation. The limit of detection for pRLX was around 0.005ng/mL (~ 0.86 fM) and the limit of confirmation was around 0.02ng/mL (~ 3.4 fM). It was observed that method sensitivity was improved at least 5-fold by using an EASY-spray column and emitter in place of the conventional ESI source. The applicability of this method was demonstrated by the identification of pRLX and its metabolites in equine plasma obtained after subcutaneous administration. To our knowledge, this is the first report of a validated mass spectrometry method for the unequivocal confirmation of pRLX in any biological fluid. Copyright © 2016 John Wiley & Sons, Ltd.

The Type B Flagellin of Hypervirulent Clostridium difficile Is Modified with Novel Sulfonated Peptidylamido-glycans

Glycosylation of flagellins is a well recognized property of many bacterial species. In this study, we describe the structural characterization of novel flagellar glycans from a number of hypervirulent strains of C. difficile. We used mass spectrometry (nano-LC-MS and MS/MS analysis) to identify a number of putative glycopeptides that carried a variety of glycoform substitutions, each of which was linked through an initial N-acetylhexosamine residue to Ser or Thr. Detailed analysis of a LLDGSSTEIR glycopeptide released by tryptic digestion, which carried two variant structures, revealed that the glycopeptide contained, in addition to carbohydrate moieties, a novel structural entity. A variety of electrospray-MS strategies using Q-TOF technology were used to define this entity, including positive and negative ion collisionally activated decomposition MS/MS, which produced unique fragmentation patterns, and high resolution accurate mass measurement to allow derivation of atomic compositions, leading to the suggestion of a taurine-containing peptidylamido-glycan structure. Finally, NMR analysis of flagellin glycopeptides provided complementary information. The glycan portion of the modification was assigned as α-Fuc3N-(1→3)-α-Rha-(1→2)-α-Rha3OMe-(1→3)-β-GlcNAc-(1→)Ser, and the novel capping moiety was shown to be comprised of taurine, alanine, and glycine. This is the first report of a novel O-linked sulfonated peptidylamido-glycan moiety decorating a flagellin protein.

High-resolution mass spectrometric determination of the synthetic cannabinoids MAM-2201, AM-2201, AM-2232, and their metabolites in postmortem plasma and urine by LC/Q-TOFMS.

High-resolution mass spectrometry and accurate mass measurement by liquid chromatography/quadrupole-time of flight mass spectrometry (LC/Q-TOFMS) was applied to postmortem plasma and urine specimens from an autopsy of a fatal case involving synthetic cannabinoid use, resulting in the detection of three synthetic cannabinoids: MAM-2201, AM-1220, and AM-2232. We searched for their metabolites existing in postmortem plasma or urine by LC/Q-TOFMS and were able to detect N-dealkylated metabolites, defluorinated and further oxidized metabolites of MAM-2201, and some hydroxylated metabolites. Postmortem plasma concentrations of the parent drugs, N-dealkylated metabolites, and fluorinated and further oxidized metabolites of MAM-2201 were measured, and quantitation results revealed site differences between heart and femoral postmortem plasma concentrations of parent drugs and some metabolites, suggesting postmortem redistribution of the synthetic cannabinoids and their metabolites. Quantitation results suggest that defluorination is a major metabolic pathway for MAM-2201, and N-dealkylation is a common but minor pathway for the naphthoylindole-type synthetic cannabinoids in human.

Detection of IGF-1 Protein Variants by Use of LC-MS with High-Resolution Accurate Mass in Routine Clinical Analysis

To the Editor: Low or increased serum insulin-like growth factor 1 (IGF-1)1 concentrations might indicate growth hormone (GH) deficiency or overproduction, respectively. Currently, IGF-1 is mostly measured by automated immunometric assays. However, reagent availability issues prompted us to evaluate LC-MS with high-resolution accurate mass measurement (HRAM) as an alternative (1). We developed and validated an IGF-1 LC-MS HRAM method on a Q Exactive mass spectrometer (Thermo Scientific) using 1:70 000 resolution (at m / z 200) with a mass accuracy of 5 ppm. A similar method has been published for a qTOF instrument (Agilent) (2). We compared our LC-MS-HRAM method initially in 459 patient samples with the iSYS IGF-1 automated immunoassay (IDS). This yielded a least square linear fit of LC-MS HRAM = 0.84 × iSYS + 3.5, with an r 2 of 0.966. During further studies involving 1720 samples to generate reference ranges, the 2 methods continued to be highly correlated, with a reproducible systematic bias. However, we also identified 16 outliers for which the LC-MS HRAM method gave dramatically lower results than the iSYS immunoassay …

Chemometric evaluation of Saccharomyces cerevisiae metabolic profiles using LC-MS.

A new liquid chromatography mass spectrometry (LC–MS) metabolomics strategy coupled to chemometric evaluation, including variable and biomarker selection, has been assessed as a tool to discriminate between control and stressed Saccharomyces cerevisiae yeast samples. Metabolic changes occurring during yeast culture at different temperatures (30 and 42 °C) were analysed and the complex data generated in profiling experiments were evaluated by different chemometric multivariate approaches. Multivariate curve resolution alternating least squares (MCR-ALS) was applied to full spectral scan LC–MS preprocessed data multisets arranged in augmented column-wise data matrices. The results showed that sectioning the MS-chromatograms in different windows and analysing them by MCR-ALS enabled the proper resolution of very complex coeluted chromatographic peaks. The investigation of possible relationships between MCR-ALS resolved chromatographic peak areas and culture temperature was then investigated by partial least squares discriminant analysis (PLS-DA). Selection of most relevant resolved chromatographic peaks associated to yeast culture temperature changes was achieved according to PLS-DA-Variable Importance in Projection scores. A metabolite identification workflow was developed utilizing MCR-ALS resolved pure MS spectra and high-resolution accurate mass measurements to confirm assigned structures based on entries in metabolite databases. A total of 65 metabolites were identified. A preliminary interpretation of these results indicates that the strategy described in this study can be proposed as a general tool to facilitate biomarker identification and modelling in similar untargeted metabolomic studies.

Insight into virgin olive oil secoiridoids characterization by high-resolution mass spectrometry and accurate mass measurements

With the aim to enhance characterization of virgin olive oil (VOO), high resolution mass spectrometry (HRMS) and high resolution tandem mass spectrometry (HRMS/MS), in positive and negative electrospray ionization (ESI) modes, coupled to fused-core reverse phase chromatography, were applied to distinct VOO phenolic extracts after the optimization of chromatographic conditions, ESI and fragmentation parameters. HRMS, but also HRMS/MS resulted fundamental to progress in secoiridoids structural elucidation. The former revealed that the secoiridoid composition of VOO was far more complicated than previously reported, while the latter helped clarify product ion elemental composition allowing new fragmentations, in addition to those reported in the literature, to be put forward. In particular, for the first time, different product ions with the same nominal mass were unequivocally identified in the spectra of secoiridoid compounds, confirming the greater capacity of HRMS/MS to clarify structure than low-resolution MS. Furthermore, and differing from previous studies, the multiple isomers of the main VOO secoiridoids could be differentiated on the basis of their HR product ion spectra in positive mode.

Mass Spectral Fragmentation Studies of Coumarin-Type Compounds Using GC High-Resolution MS

The present study was undertaken to investigate fragmentation of a set of thirteen coumarins that bear amino-, alkylamino-, alkyl-, fluoroalkyl-groups, and heterocyclic rings, in a high-resolution (>10,000) quadrupole time-of-flight mass spectrometer equipped with an the electron ionization source and interfaced to a gas chromatograph. We have demonstrated how our data processing software is used to generate molecular formulas from high-resolution accurate mass measurements and isotope ratios, and have shown that only with a high-resolution MS can one distinguish between the loss of CO, C2H4, or CH2N. EI mass spectra are shown for all coumarins tested here, and proposed fragmentation pathways based on the high-resolution MS data are proposed for two coumarins.

Characterization and Scope of S-layer Protein O-Glycosylation in Tannerella forsythia

Cell surface glycosylation is an important element in defining the life of pathogenic bacteria. Tannerella forsythia is a Gram-negative, anaerobic periodontal pathogen inhabiting the subgingival plaque biofilms. It is completely covered by a two-dimensional crystalline surface layer (S-layer) composed of two glycoproteins. Although the S-layer has previously been shown to delay the bacterium's recognition by the innate immune system, we characterize here the S-layer protein O-glycosylation as a potential virulence factor. The T. forsythia S-layer glycan was elucidated by a combination of electrospray ionization-tandem mass spectrometry and nuclear magnetic resonance spectroscopy as an oligosaccharide with the structure 4-Me-β-ManpNAcCONH(2)-(1→3)-[Pse5Am7Gc-(2→4)-]-β-ManpNAcA-(1→4)-[4-Me-α-Galp-(1→2)-]-α-Fucp-(1→4)-[-α-Xylp-(1→3)-]-β-GlcpA-(1→3)-[-β-Digp-(1→2)-]-α-Galp, which is O-glycosidically linked to distinct serine and threonine residues within the three-amino acid motif (D)(S/T)(A/I/L/M/T/V) on either S-layer protein. This S-layer glycan obviously impacts the life style of T. forsythia because increased biofilm formation of an UDP-N-acetylmannosaminuronic acid dehydrogenase mutant can be correlated with the presence of truncated S-layer glycans. We found that several other proteins of T. forsythia are modified with that specific oligosaccharide. Proteomics identified two of them as being among previously classified antigenic outer membrane proteins that are up-regulated under biofilm conditions, in addition to two predicted antigenic lipoproteins. Theoretical analysis of the S-layer O-glycosylation of T. forsythia indicates the involvement of a 6.8-kb gene locus that is conserved among different bacteria from the Bacteroidetes phylum. Together, these findings reveal the presence of a protein O-glycosylation system in T. forsythia that is essential for creating a rich glycoproteome pinpointing a possible relevance for the virulence of this bacterium.

Profiling the indole alkaloids in yohimbe bark with ultra‐performance liquid chromatography coupled with ion mobility quadrupole time‐of‐flight mass spectrometry

An ultra-performance liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry (UPLC/IM-QTOF-MS) method was developed for profiling the indole alkaloids in yohimbe bark. Many indole alkaloids with the yohimbine or ajmalicine core structure, plus methylated, oxidized and reduced species, were characterized. Common fragments and mass differences are described. It was shown that the use of IMS could provide another molecular descriptor, i.e. molecular shape by rotationally averaged collision cross-section; this is of great value for identification of constituents when reference materials are usually not available. Using the combination of high resolution (~40000) accurate mass measurement with time-aligned parallel (TAP) fragmentation, MS(E) (where E represents collision energy), ion mobility mass spectrometry (IMS) and UPLC chromatography, a total 55 indole alkaloids were characterized and a few new indole alkaloids are reported for the first time.

Overcoming the Genotoxicity of a Pyrrolidine Substituted Arylindenopyrimidine As a Potent Dual Adenosine A2A/A1 Antagonist by Minimizing Bioactivation to an Iminium Ion Reactive Intermediate

2-Amino-4-phenyl-8-pyrrolidin-1-ylmethyl-indeno[1,2-d]pyrimidin-5-one (1) is a novel and potent selective dual A(2A)/A(1) adenosine receptor antagonist from the arylindenopyrimidine series that was determined to be genotoxic in both the Ames and Mouse Lymphoma L5178Y assays only following metabolic activation. Compound 1 was identified as a frame-shift mutagen in Salmonella typhimurium tester strain TA1537 as indicated by a significant dose-dependent increase in revertant colonies as compared to the vehicle control. The metabolic activation-dependent irreversible covalent binding of radioactivity to DNA, recovery of 1 and its enamine metabolite from acid hydrolysis of covalently modified DNA, and protection of covalent binding to DNA by both cyanide ion and methoxylamine suggest that the frame-shift mutation in TA1537 strain involved covalent binding instead of simple intercalation to DNA. Compound 1 was bioactivated to endocyclic iminium ion, aldehyde, epoxide, and α,β-unsaturated keto reactive intermediates from the detection of cyano, oxime, and glutathione conjugates by data-dependent high resolution accurate mass measurements. Collision-induced dissociation of these conjugates provided evidence for bioactivation of the pyrrolidine ring of 1. The epoxide and α,β-unsaturated keto reactive intermediates were unlikely to cause the genotoxicity of 1 because the formation of their glutathione adducts did not ameliorate the binding of compound related material to DNA. Instead, the endocyclic iminium ions and amino aldehydes were likely candidates responsible for genotoxicity based on, first, the protection afforded by both cyanide ion and methoxylamine, which reduced the potential to form covalent adducts with DNA, and, second, analogues of 1 designed with low probability to form these reactive intermediates were not genotoxic. It was concluded that 1 also had the potential to be mutagenic in humans based on observing the endocyclic iminium ion following incubation with a human liver S9 preparation and the commensurate detection of DNA adducts. An understanding of this genotoxicity mechanism supported an evidence-based approach to selectively modify the structure of 1 which resulted in analogues being synthesized that were devoid of a genotoxic liability. In addition, potency and selectivity against both adenosine A(2A) and A(1) receptors were maintained.