Secondary Electrospray Ionization Research Articles

Metabolic trajectories of diabetic ketoacidosis onset described by breath analysis.

This feasibility study aimed to investigate the use of exhaled breath analysis to capture and quantify relative changes of metabolites during resolution of acute diabetic ketoacidosis under insulin and rehydration therapy. Breath analysis was conducted on 30 patients of which 5 with DKA. They inflated Nalophan bags, and their metabolic content was subsequently interrogated by secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS). SESI-HRMS analysis showed that acetone, pyruvate, and acetoacetate, which are well known to be altered in DKA, were readily detectable in breath of participants with DKA. In addition, a total of 665 mass spectral features were found to significantly correlate with base excess and prompt metabolic trajectories toward an in-control state as they progress toward homeostasis. This study provides proof-of-principle for using exhaled breath analysis in a real ICU setting for DKA monitoring. This non-invasive new technology provides new insights and a more comprehensive overview of the effect of insulin and rehydration during DKA treatment.

Abstract 4433: Secondary electrospray ionization mass spectrometry for volatile analysis of lung cancer

Abstract Lung cancer remains a leading cause of cancer-related mortality globally, necessitating innovative approaches for early detection and improved understanding of its metabolic landscape and consequences. Secondary Electrospray Ionization Mass Spectrometry (SESI-MS), an innovative analytical technique, has emerged as a powerful tool for the analysis of volatile organic compounds (VOCs) associated with various diseases, from metabolic disorders to bacterial infections. In this proof-of-concept study, we first utilized SESI-MS analyses of four representative lung cancer cells of both non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) to demonstrate its capability to profile VOCs released by lung cancer cells. The technique allows for real-time, non-invasive analysis of the headspace above cultured cells, enabling the detection of unique VOC signatures associated with different lung cancer subtypes. Meanwhile, SESI-MS also facilitated the discrimination specific cells based on distinct VOC patterns. Furthermore, we also performed cisplatin treatment experiments and observed the metabolic signature shift post drug treatment. These results collectively offered potential applications in early diagnosis and personalized treatment strategies. Next, we continued with in vivo investigations to further extend the utility of SESI-MS in lung cancer research. Animal models, mimicking human lung cancer pathophysiology, have been subjected to SESI-MS analysis to identify volatile biomarkers indicative of tumor development and progression. Several representative cancer drug treatments were performed to the lung cancer mice model, and the drug-induced responses were also monitored via SESI-MS breath analyses. The in vivo SESI-MS approach detected many interesting breath VOC features that could provide valuable insights into the dynamic changes in VOC profiles during various stages of lung cancer, and therefore has the potential to offer a deeper understanding of the disease's metabolic alterations and potential avenues for targeted therapeutic interventions. In conclusion, SESI-MS-based volatile analysis presents a robust and versatile approach for investigating lung cancer, and our ongoing work has the potential to bridge the gap between in vitro and in vivo models for better translational research outcomes. The detection of unique VOC signatures associated with lung cancer cells and tumors holds significant promise for early detection, subtype classification, and monitoring of treatment responses. As technology continues to advance, SESI-MS stands poised to play a pivotal role in transforming the landscape of lung cancer diagnostics and research. Citation Format: Jiangjiang (Chris) Zhu, Fouad Choueiry. Secondary electrospray ionization mass spectrometry for volatile analysis of lung cancer [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 4433.

Alternative electrolyte solutions for untargeted breath metabolomics using secondary-electrospray ionization high-resolution mass spectrometry.

Secondary-electrospray ionization (SESI) coupled with high-resolution mass spectrometry is a powerful tool for the discovery of biomarkers in exhaled breath. A primary electrospray consisting of aqueous formic acid (FA) is currently used to charge the volatile organic compounds in breath. To investigate whether alternate electrospray compositions could enable different metabolite coverage and sensitivities, the electrospray dopants NaI and AgNO3 were tested. In a proof-of-principle manner, the exhaled breath of one subject was analyzed repeatedly with different electrospray solutions and with the help of a spectral stitching technique. Capillary diameter and position were optimized to achieve proper detection of exhaled breath. The detected features were then compared using formula annotation. Using an evaporation-based gas standard system, the signal response of the different solutions was probed. Principal component analysis revealed a substantial difference in features detected with AgNO3 . With silver, more sulfur-containing features and more unsaturated hydrocarbon compounds were detected. Furthermore, more primary amines were potentially ionized, as indicated by van Krewelen diagrams. In total, twice as many features were unique to AgNO3 than for other electrospray dopants. Using gas standards at known concentrations, the high sensitivity of FA as a dopant was demonstrated but also indicated alternate sensitivities of the other electrospray solutions. This work demonstrated the potential of AgNO3 as a complementary dopant for further biomarker discovery in SESI-based breath analysis.

Advances in secondary electrospray ionization for breath analysis and volatilomics

The measurement of volatile organic compounds (VOCs) originating from organisms allows continuous monitoring and a unique insight into the metabolism. One method offering the sensitivity to detect these VOCs is secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS). SESI was derived from electrospray ionization (ESI) and has found widespread application in clinical research and monitoring of animals. This review discusses the technical aspects behind SESI, the advancements, and the technical hurdles faced. Additionally, the recent advances in the applications of SESI in human and animal-centered research are presented.

Real-time breath analysis towards a healthy human breath profile.

The direct analysis of molecules contained within human breath has had significant implications for clinical and diagnostic applications in recent decades. However, attempts to compare one study to another or to reproduce previous work are hampered by: variability between sampling methodologies, human phenotypic variability, complex interactions between compounds within breath, and confounding signals from comorbidities. Towards this end, we have endeavored to create an averaged healthy human 'profile' against which follow-on studies might be compared. Through the use of direct secondary electrospray ionization combined with a high-resolution mass spectrometry and in-house bioinformatics pipeline, we seek to curate an average healthy human profile for breath and use this model to distinguish differences inter- and intra-day for human volunteers. Breath samples were significantly different in PERMANOVA analysis and ANOSIM analysis based on Time of Day, Participant ID, Date of Sample, Sex of Participant, and Age of Participant (p< 0.001). Optimal binning analysis identify strong associations between specific features and variables. These include 227 breath features identified as unique identifiers for 28 of the 31 participants. Four signals were identified to be strongly associated with female participants and one with male participants. A total of 37 signals were identified to be strongly associated with the time-of-day samples were taken. Threshold indicator taxa analysis indicated a shift in significant breath features across the age gradient of participants with peak disruption of breath metabolites occurring at around age 32. Forty-eight features were identified after filtering from which a healthy human breath profile for all participants was created.

Secondary-Electrospray Ionization Mass Spectrometry-Based Online Analyses of Mouse Volatilome Uncover Gut Microbiome-Dictated Metabolic Changes in the Host.

The symbiotic relationship between the gut microbial population is capable of regulating numerous aspects of host physiology, including metabolism. Bacteria can modulate the metabolic processes of the host by feeding on nutritional components within the lumen and releasing bioactive components into circulation. Endogenous volatile organic compound (VOC) synthesis is dependent on the availability of precursors found in mammalian metabolism. Herein, we report that microbial-mediated metabolic influences can alter the host volatilome and the prominent volatile changes can be uncovered by a novel volatile analysis technique named secondary electrospray ionization mass spectrometry. Mice were subjected to an antibiotic cocktail to deplete the microbiome and then inoculated with either single strain bacteria or fecal matter transplantation (FMT) to replete the microbial population in the gut. VOC sampling was achieved by using an advanced secondary electrospray ionization (SESI) source that directly mounted onto a Thermo Q-Exactive high-resolution mass spectrometer (HRMS). A principal component analysis summarizing the volatile profiles of the mice revealed independent clustering of each strain of the FMT-inoculated groups, suggesting unique volatile profiles. The Mummichog algorithm uncovered phenylalanine metabolism as a significantly altered metabolic profile in the volatilome of the microbiome-repleted mice. Our results indicated that the systemic metabolic changes incurred by the host are translated to unique volatile profiles correlated to the diversity of the microbial population colonized within the host. It is thus possible to take advantage of SESI-HRMS-based platforms for noninvasive screening of VOCs to determine the contribution of various microbial colonization within human gut that may impact host health.

Elucidating the Role of Ion Suppression in Secondary Electrospray Ionization.

Ion suppression is a known matrix effect in electrospray ionization (ESI), ambient pressure chemical ionization (APCI), and desorption electrospray ionization (DESI), but its characterization in secondary electrospray ionization (SESI) is lacking. A thorough understanding of this effect is crucial for quantitative applications of SESI, such as breath analysis. In this study, gas standards were generated by using an evaporation-based system to assess the susceptibility and suppression potential of acetone, deuterated acetone, deuterated acetic acid, and pyridine. Gas-phase effects were found to dominate ion suppression, with pyridine exhibiting the most significant suppressive effect, which is potentially linked to its gas-phase basicity. The impact of increased acetone levels on the volatiles from exhaled breath condensate was also examined. In humid conditions, a noticeable decrease in intensity of approximately 30% was observed for several features at an acetone concentration of 1 ppm. Considering that this concentration is expected for breath analysis, it becomes crucial to account for this effect when SESI is utilized to quantitatively determine specific compounds.

Freshness evaluation of grass carp by volatile biogenic amines monitored by secondary electrospray ionization ion mobility spectrometry

In this paper, a secondary electrospray ionization ion mobility spectrometry (SESI-IMS) method using ammonium acerate was evaluated for rapid determination of the various volatile biogenic amine contents in grass carp (Ctenopharyngodon idella). The ammonium ion generated by electrospray ionization source functions as reactant ion to react with volatile biogenic amines in gas phase and separated in the drift tube. The SESI-IMS method requires minimum sample preparation and minimum interferences, and volatile biogenic amines show their unique drift times without overlapping. The content of volatile biogenic amines demonstrates good correlations with storage times at 4 ℃ and 8 ℃ cold storage. It is shown that trimethylamine and putrescine are the dominant biogenic amines during the cold storage process. Other freshness indicates including sensory evaluation, hardness, pH and total volatile basic nitrogen (TVB-N) also show good consistency with volatile biogenic amines. The developed method is a rapid, simple, and sensitive method for the determination of volatile biogenic amines in grass carp.

Prediction of systemic free and total valproic acid by off-line analysis of exhaled breath in epileptic children and adolescents.

Therapeutic drug monitoring (TDM) of medications with a narrow therapeutic window is a common clinical practice to minimize toxic effects and maximize clinical outcomes. Routine analyses rely on the quantification of systemic blood concentrations of drugs. Alternative matrices such as exhaled breath are appealing because of their inherent non-invasive nature. This is especially the case for pediatric patients. We have recently showcased the possibility of predicting systemic concentrations of valproic acid (VPA), an anti-seizure medication by real-time breath analysis in two real clinical settings. This approach, however, comes with the limitation of the patients having to physically exhale into the mass spectrometer. This restricts the possibility of sampling from patients not capable or available to exhale into the mass spectrometer located on the hospital premises. In this work, we developed an alternative method to overcome this limitation by collecting the breath samples in customized bags and subsequently analyzing them by secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS). A total ofn= 40 patients (mean ± SD, 11.5 ± 3.5 y.o.) diagnosed with epilepsy and taking VPA were included in this study. The patients underwent three measurements: (i) serum concentrations of total and free VPA, (ii) real-time breath analysis and (iii) off-line analysis of exhaled breath collected in bags. The agreement between the real-time and the off-line breath analysis methods was evaluated using Lin's concordance correlation coefficient (CCC). CCC was computed for ten mass spectral predictors of VPA concentrations. Lin's CCC was >0.6 for all VPA-associated features, except for two low-signal intensity isotopic peaks. Finally, free and total serum VPA concentrations were predicted by cross validating the off-line data set. Support vector machine algorithms provided the most accurate predictions with a root mean square error of cross validation of 29.0 ± 7.4 mg l-1and 3.9 ± 1.4 mg l-1for total and free VPA (mean ± SD), respectively. As a secondary analysis, we explored whether exhaled metabolites previously associated with side-effects and response to medication could be rendered by the off-line analysis method. We found that five features associated with side effects showed a CCC > 0.6, whereas none of the drug response-associated peaks reached this cut-off. We conclude that the clinically relevant free fraction of VPA can be predicted by this combination of off-line breath collection with rapid SESI-HRMS analysis. This opens new possibilities for breath based TDM in clinical settings.

Formation of nitrogen-containing gas phase products from the heterogeneous (photo)reaction of NO2 with gallic acid

Heterogeneous reaction of gas phase NO2 with atmospheric humic-like substances (HULIS) is potentially an important source of volatile organic compounds (VOCs) including nitrogen (N)-containing compounds, a class of brown carbon of emerging importance. However, the role of ubiquitous water-soluble aerosol components in this multiphase chemistry, namely nitrate and iron ions, remains largely unexplored. Here, we used secondary electrospray ionization ultrahigh-resolution mass spectrometry for real-time measurements of VOCs formed during the heterogeneous reaction of gas phase NO2 with a solution containing gallic acid (GA) as a proxy of HULIS at pH 5 relevant for moderately acidic aerosol particles. Results showed that the number of detected N-containing organic compounds largely increased from 4 during the NO2 reaction with GA in the absence of nitrate and iron ions to 55 in the presence of nitrate and iron ions. The N-containing compounds have reduced nitrogen functional groups, namely amines, imines and imides. These results suggest that the number of N-containing compounds is significantly higher in deliquescent aerosol particles due to the influence of relatively higher ionic strength from nitrate ions and complexation/redox reactivity of iron cations compared to that in the dilute aqueous phase representative of cloud, fog, and rain water.

Direct mass spectrometry analysis of exhaled human breath in real-time.

The molecular composition of exhaled human breath can reflect various physiological and pathological conditions. Considerable progress has been achieved over the past decade in real-time analysis of exhaled human breath using direct mass spectrometry methods, including selected ion flow tube mass spectrometry, proton transfer reaction mass spectrometry, extractive electrospray ionization mass spectrometry, secondary electrospray ionization mass spectrometry, acetone-assisted negative photoionization mass spectrometry, atmospheric pressure photoionization mass spectrometry, and low-pressure photoionization mass spectrometry. Here, recent developments in direct mass spectrometry analysis of exhaled human breath are reviewed with regard to analytical performance (chemical sensitivity, selectivity, quantitative capabilities) and applications of the developed methods in disease diagnosis, targeted molecular detection, and real-time metabolic monitoring.

Exhaled volatile fatty acids, ruminal methane emission, and their diurnal patterns in lactating dairy cows

To date, the commonly used methods to assess rumen fermentation are invasive. Exhaled breath contains hundreds of volatile organic compounds (VOC) that can reflect animal physiological processes. In the present study, for the first time, we aimed to use a noninvasive metabolomics approach based on high-resolution mass spectrometry to identify rumen fermentation parameters in dairy cows. Enteric methane (CH4) production from 7 lactating cows was measured 8 times over 3 consecutive days using the GreenFeed system (C-Lock Technology Inc.). Simultaneously, exhalome samples were collected in Tedlar gas sampling bags and analyzed offline using a secondary electrospray ionization high-resolution mass spectrometry system. In total, 1,298 features were detected, among them targeted exhaled volatile fatty acids (eVFA; i.e., acetate, propionate, butyrate), which were putatively annotated using their exact mass-to-charge ratio. The intensity of eVFA, in particular acetate, increased immediately after feeding and followed a similar pattern to that observed for ruminal CH4 production. The average total eVFA concentration was 35.5 count per second (CPS), and among the individual eVFA, acetate had the greatest concentration, averaging 21.3 CPS, followed by propionate at 11.5 CPS, and butyrate at 2.67 CPS. Further, exhaled acetate was on average the most abundant of the individual eVFA at around 59.3%, followed by 32.5 and 7.9% of the total eVFA for propionate and butyrate, respectively. This corresponds well with the previously reported proportions of these VFA in the rumen. The diurnal patterns of ruminal CH4 emission and individual eVFA were characterized using a linear mixed model with cosine function fit. The model characterized similar diurnal patterns for eVFA and ruminal CH4 and H2 production. Regarding the diurnal patterns of eVFA, the phase (time of peak) of butyrate occurred first, followed by that of acetate and propionate. Importantly, the phase of total eVFA occurred around 1 h before that of ruminal CH4. This corresponds well with existing data on the relationship between rumen VFA production and CH4 formation. Results from the present study revealed a great potential to assess the rumen fermentation of dairy cows using exhaled metabolites as a noninvasive proxy for rumen VFA. Further validation, with comparisons to rumen fluid, and establishment of the proposed method are required.

Online breath analysis with SESI/HRMS for metabolic signatures in children with allergic asthma.

Introduction: There is a need to improve the diagnosis and management of pediatric asthma. Breath analysis aims to address this by non-invasively assessing altered metabolism and disease-associated processes. Our goal was to identify exhaled metabolic signatures that distinguish children with allergic asthma from healthy controls using secondary electrospray ionization high-resolution mass spectrometry (SESI/HRMS) in a cross-sectional observational study. Methods: Breath analysis was performed with SESI/HRMS. Significant differentially expressed mass-to-charge features in breath were extracted using the empirical Bayes moderated t-statistics test. Corresponding molecules were putatively annotated by tandem mass spectrometry database matching and pathway analysis. Results: 48 allergic asthmatics and 56 healthy controls were included in the study. Among 375 significant mass-to-charge features, 134 were putatively identified. Many of these could be grouped to metabolites of common pathways or chemical families. We found several pathways that are well-represented by the significant metabolites, for example, lysine degradation elevated and two arginine pathways downregulated in the asthmatic group. Assessing the ability of breath profiles to classify samples as asthmatic or healthy with supervised machine learning in a 10 times repeated 10-fold cross-validation revealed an area under the receiver operating characteristic curve of 0.83. Discussion: For the first time, a large number of breath-derived metabolites that discriminate children with allergic asthma from healthy controls were identified by online breath analysis. Many are linked to well-described metabolic pathways and chemical families involved in pathophysiological processes of asthma. Furthermore, a subset of these volatile organic compounds showed high potential for clinical diagnostic applications.

Different reactivities of H3 O+ (H2 O)n with unsaturated and saturated aldehydes: ligand-switching reactions govern the quantitative analytical sensitivity of SESI-MS.

The detection sensitivity of secondary electrospray ionisation mass spectrometry (SESI-MS) is much lower for saturated aldehydes than for unsaturated aldehydes. This needs to be understood in terms of gas phase ion-molecule reaction kinetics and energetics to make SESI-MS analytically more quantitative. Parallel SESI-MS and selected ion flow tube mass spectrometry (SIFT-MS) analyses were carried out of air containing variable accurately determined concentrations of saturated (C5, pentanal; C7, heptanal; C8 octanal) and unsaturated (C5, 2-pentenal; C7, 2-heptenal; C8, 2-octenal) aldehyde vapours. The influence of the source gas humidity and the ion transfer capillary temperature, 250 and 300°C, in a commercial SESI-MS instrument was explored. Separate experiments were carried out using SIFT to determine the rate coefficients, k73 , for the ligand-switching reactions of the H3 O+ (H2 O)3 ions with the six aldehydes. The relative slopes of the plots of SESI-MS ion signal against SIFT-MS concentration were interpreted as the relative SESI-MS sensitivities for these six compounds. The sensitivities for the unsaturated aldehydes were 20 to 60 times greater than for the corresponding C5, C7 and C8 saturated aldehydes. Additionally, the SIFT experiments revealed that the measured k73 are three or four times greater for the unsaturated than for the saturated aldehydes. The trends in SESI-MS sensitivities are rationally explained by differences in the rates of the ligand-switching reactions, which are justified by theoretically calculated equilibrium rate constants derived from thermochemical density functional theory (DFT) calculations of Gibb's free energy changes. The humidity of SESI gas thus favours the reverse reactions of the saturated aldehyde analyte ions, effectively suppressing their signals in contrast to their unsaturated counterparts.

UHPLC-MS/MS-Based Identity Confirmation of Amino Acids Involved in Response to and Side Effects from Antiseizure Medications.

Real-time breath analysis using secondary electrospray ionization coupled with high-resolution mass spectrometry is a fast and noninvasive method to access the metabolic state of a person. However, it lacks the ability to unequivocally assign mass spectral features to compounds due to the absence of chromatographic separation. This can be overcomed by using exhaled breath condensate and conventional liquid chromatography-mass spectrometry (LC-MS) systems. In this study, to the best of our knowledge, we confirm for the first time the presence of six amino acids (GABA, Oxo-Pro, Asp, Gln, Glu, and Tyr) previously reported to be involved in response to and side effects from antiseizure medications in exhaled breath condensate and by extension in exhaled human breath. Raw data are publicly available at MetaboLights with the accession number MTBLS6760.

Miniaturized Protein Digestion Using Acoustic Levitation with Online High Resolution Mass Spectrometry.

The combination of acoustically levitated droplets, mid-IR laser evaporation, and subsequent post-ionization by secondary electrospray ionization was applied for monitoring the enzymatic digestion of various proteins. Acoustically levitated droplets are an ideal, wall-free model reactor, readily allowing compartmentalized microfluidic trypsin digestions. Time-resolved interrogation of the droplets yielded real-time information on the progress of the reaction and thus provided insights into reaction kinetics. After 30 min of digestion in the acoustic levitator, the obtained protein sequence coverages were identical to the reference overnight digestions. Importantly, our results clearly demonstrate that the applied experimental setup can be used for the real-time investigation of chemical reactions. Furthermore, the described methodology only uses a fraction of the typically applied amounts of solvent, analyte, and trypsin. Thus, the results exemplify the use of acoustic levitation as a green analytical chemistry alternative to the currently used batch reactions.

Database-assisted, globally optimized targeted secondary electrospray ionization high resolution mass spectrometry (dGOT-SESI-HRMS) and spectral stitching enhanced volatilomics analysis of bacterial metabolites.

Secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) is an innovative analytical technique for the rapid and non-invasive analysis of volatile organic compounds (VOCs). However, compound annotation and ion suppression in the SESI source has hindered feature detection, stability and reproducibility of SESI-HRMS in untargeted volatilomics. To address this, we have developed and optimized a novel pseudo-targeted approach, database-assisted globally optimized targeted (dGOT)-SESI-HRMS using the microbial-VOC (mVOC) database, and spectral stitching methods to enhance metabolite detection in headspace of anaerobic bacterial cultures. Headspace volatiles from representative bacteria strains were assessed using full scan with data dependent acquisition (DDA), conventional globally optimized targeted (GOT) method, and spectral stitching supported dGOT experiments based on a MS peaks list derived from mVOC. Our results indicate that spectral stitching supported dGOT-SESI-HRMS can proportionally fragment peaks with respect to different analysis windows, with a total of 109 VOCs fragmented from 306 targeted compounds. Of the collected spectra, 88 features were confirmed as culture derived volatiles with respect to media blanks. Annotation was also achieved with a total of 25 unique volatiles referenced to standard databases allowing for biological interpretation. Principal component analysis (PCA) summarizing the headspace volatile demonstrated improved separation of clusters when data was acquired using the dGOT method. Collectively, our dGOT-SESI-HRMS method afforded robust capability of capturing unique VOC profiles from different bacterial strains and culture conditions when compared to conventional GOT and DDA modes, suggesting the newly developed approach can serve as a more reliable analytical method for the sensitive monitoring of gut microbial metabolism.

Analysis of volatile short-chain fatty acids in the gas phase using secondary electrospray ionization coupled to mass spectrometry.

Quantification of metabolites present within exhaled breath is a major challenge for on-line breath analysis. It is also important for gauging the analytical performance, accuracy, reproducibility, reliability, and stability of the measuring technology. Short-chain fatty acids (SCFAs) are of high interest for nutrition and health. Their quantification enables a deep mechanistic understanding of a wide range of biological processes and metabolic pathways, while their high volatility makes them an attractive target for breath analysis. This article reports, for the first time, the development and testing of a modular, dynamic vapor generator for the qualitative and quantitative analysis of volatile SCFAs in the gaseous phase using a secondary electrospray ionization (SESI) source coupled to a high-resolution mass spectrometer. Representative compounds tested included acetic acid, propionic acid, butyric acid, pentanoic acid and hexanoic acid. Gas-phase experiments were performed both in dry and humid (95% relative humidity) conditions from ppt to low ppb concentrations. The results obtained exhibited excellent linearity within the examined concentration range, low limits of detection and quantification down to the lower ppt area. Mixture effects were also investigated and are presented.

Gas phase H+, H3O+ and NH4+ affinities of oxygen-bearing volatile organic compounds; DFT calculations for soft chemical ionisation mass spectrometry.

Quantum chemistry calculations were performed using the density functional theory, DFT, to understand the structures and energetics of organic ions relevant to gas phase ion chemistry in soft chemical ionisation mass spectrometry analytical methods. Geometries of a range of neutral volatile organic compound molecules and ions resulting from protonation, the addition of H3O+ and the addition of NH4+ were optimised using the B3LYP hybrid DFT method. Then, the total energies and the normal mode vibrational frequencies were determined, and the total enthalpies of the neutral molecules and ions were calculated for the standard temperature and pressure. The calculations were performed for several feasible structures of each of the ions. The proton affinities of several benchmark molecules agree with the accepted values within ±4 kJ mol-1, indicating that B3LYP/6-311++G(d,p) provides chemical accuracy for oxygen-containing volatile organic compounds. It was also found that the binding energies of H3O+ and NH4+ to molecules correlate with their proton affinities. The results contribute to the understanding of ligand switching ion-molecule reactions important for secondary electrospray ionisation, SESI, and selected ion flow tube, SIFT, mass spectrometries.

A gas-phase standard delivery system for direct breath analysis

Applications for direct breath analysis by mass spectrometry (MS) are rapidly expanding. One of the more recent mass spectrometry-based approaches is secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS). Despite increasing usage, the SESI methodology still lacks standardization procedures for quality control and absolute quantification. In this study, we designed and evaluated a custom-built standard delivery system tailored for direct breath analysis. The system enables the simultaneous introduction of multiple gas-phase standard compounds into ambient MS setups in the lower parts-per-million (ppm) to parts-per-billion (ppb) range. To best mimic exhaled breath, the gas flow can be heated (37 °C–40 °C) and humidified (up to 98% relative humidity). Inter-laboratory comparison of the system included various SESI-HRMS setups, i.e. an Orbitrap and a quadrupole time-of-flight mass spectrometer (QTOF), and using both single- as well as multi-component standards. This revealed highly stable and reproducible performances with between-run variation <19% and within-run variation <20%. Independent calibration runs demonstrated high accuracy (96%–111%) and precision (>95%) for the single-compound standard acetone, while compound-specific performances were obtained for the multi-component standard. Similarly, the sensitivity varied for different compounds within the multi-component standard across all SESI-Orbitrap and -QTOF setups, yielding limits of detections from 3.1 ppb (for p-xylene) to 0.05 ppb (for 1,8-cineol). Routinely applying the standard system throughout several weeks, allowed us to monitor instrument stability and to identify technical outliers in exhaled breath measurements. Such routine deployment of standards would significantly improve data quality and comparability, which is especially important in longitudinal and multi-center studies. Furthermore, performance validation of the system demonstrated its suitability for reliable absolute quantification while it illustrated compound-dependent behavior for SESI.