Real Time Mass Spectrometry Research Articles

Characterization of novel mutations involved in the development of resistance to colistin in Salmonella isolates from retail pork in Shanghai, China.

Salmonella is an important foodborne pathogen that poses a significant threat to food safety. This study aims to assess the prevalence, genomic features, and colistin-resistant mechanisms of Salmonella isolates collected from 118 retail pork samples from January 2021 to January 2022 in Shanghai, China. Overall, 46 (39.0%, 46/118) Salmonella isolates were collected, which were identified as 12 serotypes by genomic analysis, including Salmonella Typhimurium (n=17) and Salmonella London (n=6). Antimicrobial resistance profiling revealed that the resistance rate of these isolates to colistin was 13.0% (6/46), while 60.9% (28/46) exhibited multidrug-resistant. It was found that there were 51 distinct antimicrobial resistance genes in these 46 isolates, which were predominantly associated with resistance to aminoglycosides, fluoroquinolones, and β-lactams. More importantly, among six colistin-resistant isolates, two isolates (Salmonella Schwarzengrund and Salmonella Indiana) were found to carry the mcr-1 gene. The mechanism of resistance in the remaining four colistin-resistant isolates was further studied, and it was found that there were nine amino acid substitutions in PmrAB. It was demonstrated by site-directed mutagenesis that novel substitutions G53W in PmrA and I83V in PmrB led to colistin resistance in Salmonella (MIC=2 or 4μg/mL). Analysis results by real-time quantitative PCR and mass spectrometry indicated that the mutants PmrAG53W and PmrBI83V displayed higher expression levels of the gene pmrE than in the parental strain. This upregulation resulted in an increase in the production of 4-amino-4-deoxy-l-arabinose (L-Ara4N) that modified lipid A, thereby conferring resistance to colistin. These findings demonstrated that there was a high prevalence of MDR Salmonella isolates in retail pork in Shanghai, and the substitution G53W in PmrA and I83V in PmrB were independent factors contributing to the development of resistance to colistin in Salmonella via modification of lipid A with L-Ara4N.

Wood Species Differentiation: A Comparative Study of Direct Analysis in Real-Time and Chromatography Mass Spectrometry

This study reports for the first time the fingerprinting extractives analysis of the indigenous wood species of Podocarpus totara from New Zealand, Eucalyptus saligna from Australia and Pinus radiata imported from California, USA and grown in New Zealand. We evaluated the use of analytical techniques for wood species discrimination. We compared the chemical fingerprinting of extractive compounds obtained using traditional chromatographic techniques with direct analysis in real-time–time of flight-mass spectrometry (DART-TOF-MS) with the auxiliary of chemometrics and principal component analysis. The traditional wet chemistry analysis of wood extracts provided a comprehensive characterisation of all extractive components. However, the more eco-friendly, sustainable and faster DART-TOF-MS technique effectively distinguished between wood species when heartwood and sapwood samples were combined. Notably, neither wet chemistry nor DART-TOF-MS could clearly differentiate between heartwood and sapwood within the same wood species. DART-TOF-MS analysis demonstrates potential as a reliable quality control tool for identifying wood species necessary in commercial and timber trading markets as well as for detecting the illicit trade of counterfeit wood products.

Detection of carob flour in cocoa powder by direct analysis in real time-mass spectrometry (DART-MS)

Cocoa is a high value product and therefore a potential target for economic adulteration with less expensive ingredients. Carob flour is less expensive than cocoa powder and is frequently cited as a potential cocoa substitute. While carob has legitimate uses as a cocoa replacement, these characteristics also make it a potential adulterant of cocoa powder. Direct analysis in real time mass spectrometry (DART-MS) is an ambient ionization MS technique that can be used to rapidly interrogate samples. Samples of cocoa powders, carob flours, and mixtures of the two were extracted with buffer and interrogated by DART-MS. The mass spectra were used to develop models to distinguish between cocoa powder and cocoa powder adulterated with carob. A principal component-linear discriminant analysis (PCA-LDA) model was used to discriminate between cocoa powder and cocoa powder amended with 15% carob flour. The accuracy using internal validation was 100%. Using an external validation dataset, the accuracy, precision, and recall were 96.0%, 94.8%, and 97.3%, respectively. These results demonstrate that DART-MS can be used to discriminate between cocoa powder and cocoa powder adulterated with 15% carob.

Fingerprinting the emissions of volatile organic compounds emitted from the cooking of oils, herbs, and spices.

Emission rates for volatile organic compounds (VOCs) have been quantified from frying, spice and herb cooking, and cooking a chicken curry, using real-time selected-ion flow-tube mass spectrometry (SIFT-MS) for controlled, laboratory-based experiments in a semi-realistic kitchen. Emissions from 7 different cooking oils were investigated during the frying of wheat flatbread (puri). These emissions were dominated by ethanol, octane, nonane and a variety of aldehydes, including acetaldehyde, heptenal and hexanal, and the average concentration of acetaldehyde (0.059-0.296 mg m-3) and hexanal (0.059-0.307 mg m-3) measured during the frying was 2-10 times higher than the recommended limits for indoor environments. Total VOC emission rates were greatest for ghee (14 mg min-1), and lowest for groundnut oil (8 mg min-1). In a second series of experiments, 16 herbs and spices were individually shallow-fried in rapeseed oil. Over 100 VOCs were identified by offline gas chromatography-mass spectrometry (GC-MS), and absolute emission rates as well as oxidant reactivity for a subset of four spices were determined. These experiments allowed distinct indoor air quality profiles to be calculated for individual oils, herbs and spices, which were used to inform and interpret more realistic cooking experiments where a full recipe of chicken curry was prepared. Total-mass VOC emissions from chicken curry were dominated by methanol (62%), monoterpenes (13%) and ethanol (10%). Additionally, a clear relationship between the cooking events and the chemical classes of VOC was observed, e.g. heating the oil (aldehydes), frying spices (monoterpenes) and adding vegetables (alcohols).

Assessing Antiparasitic Compounds Persistence in Cattle Hair by DART-MS.

This study introduces an alternative strategy for evaluating antiparasitic persistence compounds in cattle hair by Direct Analysis in Real Time Mass Spectrometry (DART-MS). The developed DART-MS method aimed to determine fenthion, chlorpyrifos, and cypermethrin in cattle hair samples. DART-MS analyses were performed in positive ion mode, and parameters related to the DART source were evaluated. The analytical performance demonstrated the efficiency of the optimized DART-MS method for fenthion, chlorpyrifos, and cypermethrin quantification in the evaluated samples, meeting criteria for precision, accuracy and limits of detection. Overall, the DART-MS method provided a fast and efficient analysis for determination of antiparasitic agents in cattle hair, which contributes to the evaluation of drug administration protocols and dosage intervals, and aids the safety and advancement of the livestock sector.

Novel DART-MS approach for rapid and environmentally friendly determination of the geographical origin of hazelnuts (Corylus avellana L.)

Direct analysis in real time mass spectrometry (DART-MS) is a novel method for the authentication of food and feed that represents a serious alternative to established methods. This study aims to analyze hazelnuts from different origins and identify potential marker metabolites using a high-resolution DART-MS platform and a non-targeted metabolomics approach. To investigate the suitability of DART-MS for authenticating the origin of foods with a high fat content, 172 hazelnut samples from 5 countries were analyzed. Data evaluation using principal component analysis (PCA) and Random Forest-based classification led to an accuracy of 93.2 %, demonstrating the high valence of the DART-MS approach for verifying the origin of hazelnuts. In addition, 16 marker metabolites were identified and revealed the importance of di- and triacylglycerols for the authentication of hazelnuts. These results demonstrate the high suitability of DART-MS based analysis as a rapid, cost-effective, and environmentally friendly approach for food authentication.

In Situ Stability Analysis of Oxygen Evolution Reaction Via Mass Spectrometry

There is a growing evidence suggesting that design principles in various fields, particularly in electrochemistry, should encompass considerations of both activity and stability. Achieving high activity alongside stability is important, yet many studies still treat these aspects as separate objectives. To overcome this limitation, establishing functional links between activity and stability based on underlying reaction mechanisms is essential. Through in situ (operando) analysis, this study aims to elucidate the interplay between activity and stability, particularly in the context of the oxygen evolution reaction, utilizing real-time mass spectrometry. By conducting comprehensive investigations, we can unveil the intricate relationships between stability and activity in electrochemical environments, thus paving the way for the design of new materials that exhibit both heightened activity and robust stability.

Accessing Fungal Contributions to the Birch Effect: Real-Time Respiration from Pore-Scale Microfluidics.

Drying and rewetting of soil stimulates soil carbon emission. The Birch effect, driven by these cycles, leads to CO2 efflux, which can be monitored using real-time mass spectrometry (RTMS). Although soil fungi retain water during droughts, their contribution to CO2 release during drying-rewetting cycles remains unclear. In this study, we present the first demonstration of integrating micromodels with RTMS to monitor the Birch effect by simulating drought and rewetting. Micromodels were inoculated with axenic fungal culture and dried to assess moisture retention. After drying, RTMS quantified CO2 release upon rewetting with H218O mixtures. Our results showed that soil fungi released CO2 upon rehydration and immediately utilized the external water source at the pore scale by generating subsequent 46CO2. This work is the first to integrate RTMS with microsystems to investigate pore-scale biogeochemistry and the involvement of fungi in the Birch effect.

Rapid quantitative analysis of semi-volatile organic compounds in indoor surface film using direct analysis in real time mass spectrometry: a case study on phthalates

Abstract. Direct analysis in real time mass spectrometry (DART-MS) has recently emerged as a promising approach for measuring semi-volatile organic compounds (SVOCs) on indoor surface films. However, its broader application in indoor environments is limited by low measurement repeatability and no separation of isomers. Herein we developed a sampling suite of indoor surface films for DART-MS analysis, optimized settings of DART to obtain higher analytical performance, and demonstrated the possibility of separating isomeric compounds using tandem mass spectrometry (MS/MS). Two pairs of isomeric phthalate esters, including di(2-ethylhexyl) phthalate (DEHP) and di-n-octyl phthalate (DnOP) and diisobutyl phthalate (DiBP) and di-n-butyl phthalate (DnBP), were used as examples for method optimization and validation. Under optimized conditions, the instrument responses for all four compounds exhibited good linearity (r>0.992) and acceptable repeatability (intraday relative standard deviation (RSD) < 11.0 %). The limits of quantification for the four phthalate esters ranged from 0.042 to 0.24 ng cm−2. The uncertainty in the separation of isomeric components using MS/MS was < 11.4 %, which is acceptable for real sample analysis. To further assess the developed method, we analyzed 10 film samples collected side by side in an occupied office. DnOP was not detected. The RSD among samples was 6.1 % for DEHP, 4.6 % for DnBP, and 10.4 % for DiBP, indicating the overall good repeatability of the collection and measurement method developed. With improved performance, the developed method increases the feasibility of the DART-MS technique for monitoring the dynamics of chemical composition of indoor surface films.

A Comparison of Molecular Techniques for Improving the Methodology in the Laboratory of Pharmacogenetics.

One of the most critical goals in healthcare is safe and effective drug therapy, which is directly related to an individual's response to treatment. Precision medicine can improve drug safety in many scenarios, including polypharmacy, and it requires the development of new genetic characterization methods. In this report, we use real-time PCR, microarray techniques, and mass spectrometry (MALDI-TOF), which allows us to compare them and identify the potential benefits of technological improvements, leading to better quality medical care. These comparative studies, as part of our pharmacogenetic Five-Step Precision Medicine (5SPM) approach, reveal the superiority of mass spectrometry over the other methods analyzed and highlight the importance of updating the laboratory's pharmacogenetic methodology to identify new variants with clinical impact.

Recovery and detection of ignitable liquid residues from the substrates by solid phase microextraction – direct analysis in real time mass spectrometry

In this study, direct analysis in real time mass spectrometry (DART-MS) was coupled to the solid phase microextraction (SPME) to extract and analyze the ignitable liquid residues (ILR) present in the sample matrices. The SPME extraction parameters, such as extraction temperature and extraction time, were optimized using a two-factor central composite design. The SPME-DART-MS setup was utilized to analyze the substrates and fire debris matrices spiked with gasoline. The results indicate that the less volatile marker compounds from gasoline were recovered from the substrates and fire debris, and their profiles matched well with the gasoline liquid samples analyzed directly by DART-MS. As expected, the effective extraction of marker compounds in gasoline required a relatively high temperature, i.e., 150 ℃. In the presence of a matrix, a higher extraction temperature and longer extraction time could benefit the extraction efficiency. The desorption of ILR on SPME fiber was performed by inserting the fiber into the DART-MS helium gas stream at 300 ℃ for 1 min with no carry-over residues being observed between successive samples. The chemical information attained with this method is typically not observed in the current GC/MS-based practice. The SPME-DART-MS was also extended to reanalyze less volatile components of ILR on substrates after the ASTM E1412 activated charcoal method, which indicates its possible application subsequent to the traditional GC/MS ILR analysis. The SPME-DART-MS has shown promise in ILR detection as an important complementary tool.

VOCs conversion in He/H2O plasma produced in a micro-capillary tube at atmospheric pressure

Abstract A non-equilibrium plasma is created in a micro-capillary quartz tube (800 µm of internal diameter), by a DC-pulsed micro-dielectric barrier discharge (micro-DBD) and the propagation of an ionisation wave, in mixtures of He/H2O/VOC at atmospheric pressure where the studied volatile organic compounds (VOCs) are representative of molecules belonging to different chemical families: alcohols (methanol, ethanol, isopropanol, tert-butanol), ketones (acetone), nitriles (acetonitrile), and aromatic hydrocarbons (toluene). The conversion efficiency of these VOCs is studied as a function of the applied voltage on the micro-DBD (or electrical energy deposited in the plasma) and of the initial concentration of the molecules in the range from 1 ppm up to 3000 ppm (depending on the molecule), with the help of high-resolution real-time mass spectrometry Fourier transform ion cyclotron resonance associated to chemical ionisation (CI-FTICR) using H3O+ as precursor ion. A variety of by-products resulting from the conversion of VOCs are identified and quantified, emphasising that the micro-capillary plasma is able to induce a complex chemical reactivity. A qualitative analysis of the involved kinetics, based on the existing literature, reveals that helium species (ions and metastable states) and radicals coming from the dissociation of the water molecules (O and OH) are the most probable candidates to explain the formation of all compounds detected by the CI-FTICR apparatus. Quenching processes of the metastable He(23S) by the VOCs, leading to the dissociation of the molecules, are suggested to explain some of the experimental results.

Hepatocyte-derived Igκ promotes HCC progression by stabilizing electron transfer flavoprotein subunit α to facilitate fatty acid β-oxidation

BackgroundLipid metabolism dysregulation is a key characteristic of hepatocellular carcinoma (HCC) onset and progression. Elevated expression of immunoglobulin (Ig), especially the Igκ free light chain with a unique Vκ4-1/Jκ3 rearrangement in cancer cells, is linked to increased malignancy and has been implicated in colon cancer tumorigenesis. However, the role of Igκ in HCC carcinogenesis remains unclear. The aim of this study was to elucidate the pivotal roles of hepatocyte-derived Igκ in HCC development.MethodsThe rearrangement sequence and expression level of hepatocyte-derived Igκ in HCC cells were determined via RT-PCR, Sanger sequencing, immunohistochemistry, and western blot analysis. The function of Igκ in HCC tumorigenesis was assessed by silencing Igκ using siRNA or gRNA in various HCC cell lines. To assess the role of Igκ in HCC pathogenesis in vivo, a mouse model with hepatocyte-specific Igκ knockout and diethylnitrosamine (DEN) and carbon tetrachloride (CCL4)-induced HCC was utilized. The molecular mechanism by which Igκ affects HCC tumorigenesis was investigated through multiomics analyses, quantitative real-time PCR, immunoprecipitation, mass spectrometry, immunofluorescence, and metabolite detection.ResultsWe confirmed that Igκ, especially Vκ4-1/Jκ3-Igκ, is highly expressed in human HCC cells. Igκ depletion inhibited HCC cell proliferation and migration in vitro, and hepatocyte-specific Igκ deficiency ameliorated HCC progression in mice with DEN and CCL4-induced HCC in vivo. Mechanistically, Vκ4-1/Jκ3-Igκ interacts with electron transfer flavoprotein subunit α (ETFA), delaying its protein degradation. Loss of Igκ led to a decrease in the expression of mitochondrial respiratory chain complexes III and IV, resulting in aberrant fatty acid β-oxidation (FAO) and lipid accumulation, which in turn inhibited HCC cell proliferation and migration.ConclusionOur findings indicate that the Igκ/ETFA axis deregulates fatty acid β-oxidation, contributing to HCC progression, which suggests that targeting fatty acid metabolism may be an effective HCC treatment strategy. The results of this study suggest that hepatocyte-derived Vκ4-1/Jκ3-Igκ may serve as a promising therapeutic target for HCC.

Bioactive Compounds and Valorization of Coffee By-Products from the Origin: A Circular Economy Model from Local Practices in Zongolica, Mexico.

The by-products of green coffee processing are rich in compounds that can be recycled for their possible use in the production of beverages, fertilizers and weed control in production areas. The objective of this work was to identify the organic and inorganic bioactive compounds of green coffee and the coffee by-products related to the production of origin, such as dried cascara (skin-pulp), parchment and silverskin (unroasted), in order to investigate the role their biomolecules may have in reuse through practices and local knowledge, not yet valued. The metabolomic profile by HPLC-ESI-HRMS of the aqueous extract of the dried cascara highlighted 93 non-volatile molecules, the highest number reported for dried cascara. They belong to groups of organic acids (12), alkaloids (5), sugars (5), fatty acids (2), diglycerides (1), amino acids (18), phospholipids (7), vitamins (5), phenolic acids (11), flavonoids (8), chlorogenic acids (17), flavones (1) and terpenes (1). For the first time, we report the use of direct analysis in real-time mass spectrometry (DART-MS) for the identification of metabolites in aqueous extracts of dried cascara, parchment, silverskin and green coffee. The DART analysis mainly showed the presence of caffeine and chlorogenic acids in all the extracts; additionally, sugar adducts and antioxidant compounds such as polyphenols were detected. The mineral content (K, Ca, P, S, Mg and Cl) by EDS spectrometry in the by-products and green coffee showed a relatively high content of K in the dried cascara and green coffee, while Ca was detected in double quantity in the silverskin. These metabolomic and mineral profile data allow enhancement of the link between the quality of green coffee and its by-products and the traditional local practices in the crop-growing area. This consolidates the community's experience in reusing by-products, thereby minimizing the impact on the environment and generating additional income for coffee growers' work, in accordance with the principles of circular economy and bioeconomy.

Improved Discrimination of Mass Spectral Isomers Using the High-Dimensional Consensus Mass Spectral Similarity Algorithm.

This study employs a high-dimensional consensus mass spectral (HDCMS) similarity scoring technique to discriminate isomers collected using an electron ionization mass spectrometer. The HDCMS method was previously introduced and applied to the discrimination of mass spectra of constitutional isomers, methamphetamine and phentermine, collected with direct analysis real-time mass spectrometry (DART-MS). The method formulates the problem of discriminating mass spectra in a mathematical Hilbert space and is hence called "high dimensional." It requires replicate mass spectra to build a Gaussian model and evaluate the inner products between these functions. The resulting measurement variability is used as a signature by which to discriminate spectra. In this work, HDCMS is tested on electron impact ionization (EI) mass spectra of 7 terpene and terpene-related (C10H16 and C10H14) isomers with experimental retention indices that differ by less than 30 and with traditional cosine similarity scores greater than 0.9, on a scale of 0 to 1, when compared with at least one other compound in the test set. Using identical instrument parameters, 15 replicate gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) spectra of each isomer were collected and separated into distinct library and query sets. The HDCMS algorithm discriminated each isomer, indicating the method's potential. Because the method requires replicate measurements, observations from a simple heuristic study of the number of replicates required to discriminate these isomers is presented. The paper concludes with a discussion of compound discrimination using HDCMS and the benefits and drawbacks of applying the method to EI-MS data.

Automated Identification of Outgassed Chemical Species Using Mass Spectrometry for Scientific Space Missions

Current and planned space exploration and life detection missions such as NASA Jet Propulsion Laboratory’s Mars 2020, Mars Sample Return, and Europa Clipper require record-low contamination levels. In this work, we develop a novel automated outgassing contamination species identification method using mass spectrometry data. Thanks to the existing knowledge of ASTM-E1559 and direct analysis in real-time mass spectrometry material testing methods, we improved species outgassing deconvolution and physical characterization techniques. We developed a brand-new combined approach that leverages both tests’ complementary insights into contaminants’ chemical identities. Namely, we rely on the Dromey–Foyster algorithm and the Seven Golden Rules of mass spectrometry analysis to narrow down the probable identified molecules by a factor of 10 compared to the sole use of ASTM-E1559. Furthermore, thanks to recent advances in machine learning spectral analysis, we developed a composite spectral score that ranks all candidate contaminants based on their similarity to the measurements. Ultimately, we validated an automated species identification algorithm on samples of NuSil CV4-2946, Dacron, and a spacecraft cable component. The techniques established in this work enable accurate estimations of outgassed contaminant species’ chemical structure, leading to a comprehensive understanding of the effects of outgassing on scientific objectives.

Direct analysis in real time mass spectrometry (DART-MS/MS) for rapid urine opioid detection in a clinical setting

Background and aimsCurrent laboratory methods for opioid detection involve an initial screening with immunoassays which offers efficient but non-specific results and a subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation which offers accurate results but requires extensive sample preparation and turnaround time. Direct Analysis in Real Time (DART) tandem mass spectrometry is evaluated as an alternative approach for accurate opioid detection with efficient sample preparation and turnaround time. Materials and methodsDART-MS/MS was optimized by testing the method with varying temperatures, operation modes, extraction methods, hydrolysis times, and vortex times. The method was evaluated for 12 opioids by testing the analytical measurement range, percent carryover, precision studies, stability, and method-to-method comparison with LC-MS/MS. ResultsDART-MS/MS shows high sensitivity and specificity for the detection of 6-acetylmorphine, codeine, hydromorphone, oxymorphone, hydrocodone, naloxone, buprenorphine, norfentanyl, and fentanyl in urine samples. However, its performance was suboptimal for norbuprenorphine, morphine and oxycodone. ConclusionIn this proof-of-concept study, DART-MS/MS is evaluated for its rapid quantitative definitive testing of opioids drugs in urine. Further research is needed to expand its application to other areas of drug testing.

Two polymorph modifications of tris(hexafluoroacetylacetonato)iron(III) revealed: is that common for other trivalent metals?

A long-standing issue about the correct identification of an important starting reagent, iron(III) hexafluoroacetylacetonate, Fe(hfac)3 (1), has been resolved. The tris-chelated mononuclear complex was found to crystallize in two polymorph modifications which can be assigned as the low-temperature (1-L) monoclinic P21/n and the high-temperature (1-H) trigonal P-3. Low-temperature polymorph 1-L was found to transform to 1-H upon sublimation at 44 °C. Two modifications are clearly distinguished by powder X-ray diffraction (PXRD), single-crystal X-ray diffraction, differential scanning calorimetry (DSC), and melting-point measurements. On the other hand, the two forms share similar characteristics in direct analysis in real-time mass spectrometry (DART-MS), attenuated total reflection (ATR) spectroscopy, and some physical properties, such as color, volatility, sensitivity, and solubility. Analysis of the literature and some of our preliminary data strongly suggest that the appearance of two polymorph modifications for trivalent metal (both transition and main group) hexafluoroacetylacetonates is a common case for several largely used complexes not yet accounted for in the crystallographic databases.

Understanding Flooding in Cu-Gde Based CO2 Electrolyzers Via Real Time Mass Spectroscopy

In the recent years, CO2 electrolyzers based on Cu-coated GDE systems became a hot topic of various research groups. As the field broadens, we are gaining better control over product selectivity, which coupled to achieving industrially relevant current densities (>300 mA/cm²), is bringing the inevitable widespread industrial adoption closer than ever. A huge limiting factor in economically viable big-scale CO2 electroreduction however, is still it´s long-term operational stability. The latter depends on two factors - catalyst stability and gas diffusion electrode´s resistance to electrowetting. In this work, we utilize a direct real time mass spectrometry approach to dive deeper into mechanism of GDE electrowetting, trying to understand what causes it and eventually make first steps in it´s prevention.

Protonation Effects on the Benzoxazine Formation Pathways and Products Distribution.

The effect of acidic media on the formation of the 3,4-dihydro-2H-3-phenyl-1,3-benzoxazine Bz is evaluated, focusing on the differentiation of intermediates and products formed by the distinct pathways observed in the presence and absence of acid. The use of real-time mass spectrometry (PSI-ESI-MS) coupled to tandem mass spectrometry and infrared multiple photon dissociation (IRMPD) allowed the differentiation of the species observed during the synthesis of benzoxazines in these different conditions. The results suggest that formic acid promotes the formation of aniline and phenol condensation products (IC and IIC) by protecting the aniline amino group and enhancing the formaldehyde electrophilicity. The results also suggest that although the presence of acid allow a more efficient potential energy landscape to be accessed, the last cyclization step for the formation of benzoxazines cannot be mediated by the protonation route intermediate (ROP Bz). Overall, the conclusions presented here provide important information about the synthesis of benzoxazines under acidic conditions, allowing the development of optimal reaction conditions.