Internal Standard Addition System for Online Breath Analysis.

Breath analysis with secondary electrospray ionization (SESI) coupled to mass spectrometry (MS) is a sensitive method for breath metabolomics. To enable quantitative assessments using SESI-MS, a system was developed to introduce controlled amounts of gases into breath samples and carry out standard addition experiments. The system combines gas standard generation through controlled evaporation, humidification, breath dilution, and standard injection with the help of mass-flow controllers. The system can also dilute breath, which affects the signal of the detected components. This response can be used to filter out contaminating compounds in an untargeted metabolomics workflow. The system's quantitative capabilities have been shown through standard addition of pyridine and butyric acid into breath in real time. This system can improve the quality and robustness of breath data.

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

RATIONALE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: This work demonstrated the potential of AgNO3 as a complementary dopant for further biomarker discovery in SESI-based breath analysis.

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.

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.

How Soft Is Secondary Electrospray Ionization?

Secondary electrospray ionization (SESI) mass spectrometry (MS) is a direct infusion technique often used for untargeted metabolomics, e.g., for online breath analysis. SESI is thought to be a soft ionization method, which is important to avoid interference from in-source fragments and to simplify compound annotation. In this work, benzylammonium ions, formed from volatile benzylamines, with known bond dissociation enthalpies were used as thermometer ions to investigate the internal energy distribution of ions that are produced by SESI. It is shown that SESI is softer than electrospray ionization (ESI), and therefore, SESI indeed qualifies as a soft ionization technique. However, we also found that the standard MS instrument settings used in the SESI community are relatively harsh. Proper soft tuning of the instrument is essential to fully benefit from the softness that SESI can provide. Moreover, there is evidence from in-source collision-induced dissociation (CID) experiments that analytes can be solvated in SESI under soft conditions, which supports a recently proposed SESI mechanism referred to as ligand switching.

Breath response following a nutritional challenge monitored by secondary electrospray ionization high-resolution mass spectrometry.

On-line breath analysis using secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS) is a sensitive method for biomarker discovery. The strengths of this technology have already been demonstrated in the clinical environment. For the first time, this study demonstrates the application of SESI-HRMS in the field of nutritional science using a standardized nutritional intervention, consisting of a high-energy shake (950 kcal, 8% protein, 35% sugar and 57% fat). Eleven subjects underwent the intervention on three separate days and their exhaled breath was monitored up to six hours postprandially. In addition, sampling was performed during equivalent fasting conditions for selected subjects. To estimate the impact of inter- and intra-individual variability, analysis of variance simultaneous component analysis was conducted, revealing that the inter-individual variability accounted for 30% of the data variation. To distinguish the effect of the intervention from fasting conditions, partial least squares discriminant analysis was performed. Candidate compound annotation was performed with pathway analysis and collision-induced dissociation (CID) experiments. Pathway analysis highlighted, among others, features associated with the metabolism of linoleate, butanoate and amino sugars. Tentative compounds annotated through CID measurements include fatty acids, amino acids, and amino acid derivatives, some of them likely derived from nutrients by the gut microbiome (e.g. propanoate, indoles), as well as organic acids from the Krebs cycle. Time-series clustering showed an overlap of observed kinetic trends with those reported previously in blood plasma.