Ricardo Vessecchi

Computational chemistry has played a key role in helping scientists understand ionization and molecular dissociation in the gas phase, which has contributed to its growing popularity among users of various mass spectrometry techniques. Early studies laid a strong foundation for applying computational methods to explore the stability and reactivity of gas‐phase ions, making these approaches more accessible and widely used across different research areas. In this work, we present a practical guide for effectively using computational chemistry in mass spectrometry studies. By comparing experimental results obtained in ESI‐MS/MS with those from quantum chemical models (DFT and composite methods) and spectra simulations (QCxMS and CFM‐ID), we aim to provide researchers with clear steps for studying organic molecules from ESI‐MS and MS/MS data and understanding the protonation in mass spectrometry. Proton affinity and gas‐phase basicities were calculated and the protonation sites were proposed. These results were compared to atomic charges, molecular orbitals, and Quantum Theory of Atoms‐in‐Molecules (QTAIM) calculations. Fragmentation mechanisms were proposed from protonated species, and the MS/MS spectra were compared to those predicted using CFM‐ID and QCxMS methods. The 2‐methyl‐1,4‐naphthoquinone analysis was used as a reference molecule for computational studies and benchmark. It is suggested the useful toolkits to describe molecular ionization/dissociation in ESI‐MS and ESI‐MS/MS studies from quantum chemical calculations by using a guide for the systematic application of computational chemistry in mass spectrometry and fragmentation mechanisms studies. Finally, an application was demonstrated for 2‐hydroxy‐3‐benzamine‐1,4‐naphthoquinone ionized in positive mode, highlighting the versatility of this guideline for studies involving more structural complexity.

Rationale: Ketoconazole (KCZ) is a clinically essential antifungal and potent CYP3A4 inhibitor. Its gas‐phase fragmentation under positive electrospray ionization tandem mass spectrometry (ESI‐MS/MS) remains poorly characterized, undermining metabolite profiling and structural identification of related azoles. Methods: We systematically mapped the fragmentation pathways of protonated KCZ using a dual‐platform strategy combining quadrupole time‐of‐flight collision‐induced dissociation (QTOF‐CID) and Orbitrap higher‐energy collisional dissociation (HCD) across a 10–60‐eV collision energy range. Mechanistic assignments and protonation sites were supported by density functional theory (DFT) calculations at B3LYP/6‐311++G(d,p) level in order to quantify thermodynamic barriers and dissociation energies. Results: Our study identified three distinct dissociation pathways: (1) piperazine ring opening driven by ketene loss (A1, m/z 489.1453) and alkylamine eliminations; (2) 1,3‐dioxolane ring cleavages (e.g., C10, m/z 277.1547); and (3) radical‐driven dissociation at the ether linker, yielding an imidazole radical cation (A28, m/z 82.0531, C ₄ H ₆ N ₂ ^•+ ). DFT calculations established a clear thermodynamic hierarchy, where even‐electron ions (e.g., A1, ΔG ≈ 19 kcal mol ⁻¹ ) prevail at low energies, whereas the radical cation (A28, ΔG ≈ 86 kcal mol ⁻¹ ) is only accessible at ≥ 40 eV. HCD conditions selectively promoted odd‐electron ion formation, revealing platform‐dependent fragmentation behavior. Conclusions: This work delivers the first comprehensive, computationally validated fragmentation map for KCZ, directly linking MS/MS observations to thermodynamic predictions and providing a robust reference for high‐confidence identification of synthetic imidazole antifungals.

Pyrimido[1,6-b]indazole represents a recently reported fused heterocyclic framework whose electronic structure remains unexplored. In this work, we present a comprehensive structural and electronic investigation of this scaffold through an integrated experimental–theoretical approach combining NMR spectroscopy, density functional theory (DFT) calculations, and single-crystal X-ray diffraction. Detailed 1H and 13C NMR analyses, supported by GIAO–DFT calculations, enabled reliable spectral assignment and revealed efficient long-range electronic communication across the rigid π-conjugated system. Single-crystal X-ray diffraction confirmed a highly planar architecture, while bond metric analysis disclosed pronounced differentiation of C–N and C–C bond lengths throughout the fused core. Quantitative correlation between experimental and computed geometrical parameters demonstrated excellent agreement, validating the computational model for probing subtle electronic effects. Comparative analysis with related aromatic systems highlights significant bond-length alternation and electronic asymmetry induced by heterocycle fusion and heteroatom incorporation. These features provide consistent evidence for a non-uniform distribution of π-electron density, supporting the description of modulated aromaticity within the tricyclic framework. In the solid state, intermolecular organization is governed by π–π stacking and dispersion-driven contacts, as supported by Hirshfeld surface analysis. Together, these findings establish pyrimido[1,6-b]indazole as a representative model for investigating electronic polarization in fused heteroaromatic systems, offering new insights into structure–property relationships beyond classical aromaticity descriptions.

The fragmentation of four deprotonated plumeran indole (PIAs), namely alkaloids aspidospermidine (1), demethoxypalosine (2), aspidocarpine (3), and aspidolimine (4) plumeran indole alkaloids (PIA), previously isolated from Aspidosperma spruceanum has been investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the negative ion mode. The fragmentation pathways have been established on the basis of accurate mass data. Our results demonstrated that the main product ions of deprotonated 1-4 result from remote hydrogen rearrangements. The most abundant product ion in the product ion spectrum of 2, 3, and 4 was the result of a ketene elimination (methylketene for 2 and 3, and a ketene for 4) directly from the precursor ion. The product ion of m/z 183 was diagnostic for compound 2, whereas the radical elimination of •CH3 occurred only for 3 and 4, which display an aromatic methoxyl group in their structures. These results indicated that ESI(–)-MS/MS could be also used for the identification of PIAs 1-4 in crude extracts using LC-ESI-MS/MS, especially when the extracts are more complex

Rationale: Dengue, an arboviral disease transmitted by the Aedes aegypti mosquito, is a major global public health problem challenge. Insecticides based on natural products can provide a good alternative to synthetic agents, as they are safer for both the environment and human health. This study evaluated the activity of geopropolis from stingless bees and Apis mellifera bees on Ae. aegypti , using mass spectrometry approaches to identify compounds with larvicidal potential against Ae. aegypti . Methods: The larvicidal activity of propolis from stingless bees and A. mellifera , as well as the Melipona quadrisfasciata geopropolis (a mixture of soil/clay and propolis), was evaluated against Ae. aegypti larvae (Rockefeller strain). ESI‐MS/MS analyses were performed using a quadrupole/time‐of‐flight mass spectrometer for all geopropolis samples, the geopropolis from Melipona quadrifasciata was also analyzed using an ion trap instrument. The ESI‐qTOF‐MS/MS data were processed in a spectral similarity network using GNPS. Molecular annotation of potential compounds was performed using the in silico tool called NAP. Gas‐phase fragmentation mechanisms were proposed in conjunction with computational chemistry studies. Silylated geopropolis samples were also analyzed by GC–MS. Results: Geopropolis from the stingless bee M. quadrifasciata caused 90% and 100% mortality in Ae. aegypti larvae after 24 and 48 h of exposure, respectively, exhibiting the highest activity. Mass spectrometry‐based molecular network approach supported the suggestion of discriminant compounds between active and inactive samples. The combination of NAP predictions with gas‐phase reactions from ESI‐MS/MS and EI‐MS data facilitated the annotation of larvicidal compounds, including diterpene resin acids, such as dehydroabietic acid and its derivatives, abietic acid, and pimaranes. Conclusion: The combination of HPLC‐MS/MS and GC–MS data suggests that diterpene resin acids contribute to the larvicidal effect of M. quadrifasciata geopropolis on Ae. aegypti , enhancing our understanding of potentially bioactive natural products against the arbovirus vector.

Star fruit belongs to the Oxalidaceae family, originally from Asia and widely distributed worldwide as a food source. Traditional knowledge advises patients with kidney problems to avoid consuming this fruit. Bioguided studies have led to the isolation of a toxin with a structural core analogous to the amino acids phenylalanine and tyrosine. Mass spectrometry (MS) is the primary technique used for analyzing biological samples, as understanding the gas-phase decomposition reactions of target analytes enhances the efficiency of these analyses. In this study, we detail the mechanisms involved in the formation of the main ion fragments of caramboxin in electrospray ionization tandem mass spectrometry (ESI-MS/MS), supported by computational chemistry and mass spectral simulations.

Rationale: Macrolides are critical antibiotics featuring a macrocyclic lactone core with deoxy sugars. Understanding their gas‐phase fragmentation is challenging but essential for improving structural elucidation in mass spectrometry, which has implications for drug discovery and development. Methods: We used electrospray ionization collision‐induced dissociation tandem mass spectrometry (ESI‐CID‐MS) combined with quantum chemical calculations to investigate the fragmentation pathways of erythromycin A and roxithromycin. This approach helps elucidate the preferred fragmentation routes influenced by protonation sites. Results: Macrolides showed similar fragmentation patterns, including sequential losses of saccharide or amino sugar units and dehydration from the macrocycle core. Multiple competitive pathways were observed, influenced by protonation sites. Computational studies confirmed the most favorable protonation sites and their impact on fragmentation, providing insights into key diagnostic product ions. Subsequent fragments involved rearrangement pathways such as alkene formation and cleavages via remote hydrogen transfers and pericyclic reactions. Conclusions: Our integrated approach offers a comprehensive understanding of macrolide fragmentation, enhancing structural elucidation and potential applications in drug development. This study advances mass spectrometry analysis of macrolides, contributing to pharmaceutical research by integrating orthogonal annotation methods and fragmentation studies.

We have investigated the gas‐phase fragmentation reactions of 11 synthetic 4‐aryl‐3,4‐dihydrocoumarins by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) on a quadrupole‐time‐of flight (Q‐TOF) hybrid mass spectrometer. We have also estimated thermochemical data for the protonated coumarins (precursor ion A) and product ion structures by computational chemistry at a B3LYP level of theory to establish the ion structures and to rationalize the fragmentation pathways. The most abundant ions in the product ion spectra of coumarins 1–11 resulted from C₈H₈O₂, CO₂, C₄H₄O₃, C₈H₁₀O₃, C₈H₈O₂, and CH₃OH eliminations through retro‐Diels–Alder (RDA) reactions, remote hydrogen rearrangements (β‐eliminations), and β‐lactone ring contraction. Although the investigated coumarins shared most of the fragmentation pathways, formation of a benzylic product ion and its corresponding tropylium ion was diagnostic of the substituents at ring C. The thermochemical data revealed that the nature and position of the substituents at ring C played a key role in the formation of this product ion and determined its relative intensity in the product ion spectrum. The results of this study contribute to knowledge of the gas‐phase ion chemistry of this important class of organic compounds.

We have investigated the gas-phase fragmentation reactions of eleven synthetic 4-aryl-3,4-dihydrocoumarins by electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a quadrupole-time-of flight (Q-TOF) hybrid mass spectrometer. We have also estimated thermochemical data for the protonated coumarins (precursor ion A) and product ion structures by computational chemistry at a B3LYP level of theory to establish the ion structures and to rationalize the fragmentation pathways. The most intense peaks in the product ion spectra of coumarins 1–11 resulted from C8H8O2, CO2, C4H4O3, C8H10O3, C8H8O2, and CH3OH eliminations through retro-Diels-Alder (RDA) reactions, remote hydrogen rearrangements (β-eliminations), and β-lactone ring contraction. Although the investigated coumarins shared most of the fragmentation pathways, formation of a benzylic product ion and its corresponding tropylium ion was diagnostic of the substituents at ring C. The thermochemical data revealed that the nature and position of the substituents at ring C played a key role in the formation of this product ion and determined the relative intensity of its peak in the product ion spectrum. The results of this study contribute to knowledge of the gas-phase ion chemistry of this important class of organic compounds.

Rationale: Oxazolines are important compounds for drug development, synthesis, and pharmaceutical applications. Interest in analyzing and developing methods to characterize reaction products from these small heterocyclics has led us to study the gas‐phase reactivity and fragmentation of seven 2‐arene‐2‐oxazolines compounds using computational chemistry combined with mass spectrometry. Method: Protonation sites were investigated using computed proton affinity, gas‐phase basicity, and some quantum chemistry descriptors of reactivity; the B3LYP/6–31+G(d,p) computational model was used. Fragmentation mechanisms were suggested by employing data from collision‐induced dissociation (CID), energy‐resolved plots from MS/MS spectra, multiple‐stage experiments, and survival‐yield method. Results: Protonation studies based on quantum theory of atoms in molecules (QTAIM) and computational thermochemistry were useful to describe the reactivity of the investigated 2‐arene‐2‐oxazolines, which can be protonated at the nitrogen atom. Three major fragmentation pathways were identified for the protonated molecules: formation of (a) benzoylium or (b) nitrilium ions through elimination of 71 and 72 u from the protonated molecules, respectively, and (c) elimination of 54 u from [M+H] ⁺ . These pathways were exploited by the density functional theory calculations combined with QTAIM studies. Conclusions: Our results can help in identifying 2‐arene‐2‐oxazoline derivatives using electrospray ionization tandem mass spectrometry (ESI‐MS/MS), which can be applied for monitoring reactions through the identified diagnostic ions (product ions). Also, we can suggest that benzoylium and nitrilium ions emerge during fragmentation under CID conditions.