Direct Mass Spectrometric Analysis of Exhaled Breath: Advances towards Clinical Application
Bettina Streckenbach
Abstract
Metabolomics, or the comprehensive study of metabolites and involved processes, provides insights into the current physiological state of humans. In the clinical setting, the detection and quantification of specific metabolites has proven to be invaluable in diagnostic testing. Herein, body samples that are non-invasively accessible are of particular interest. Exhaled breath contains metabolites than can be detected using highly sensitive technologies like mass spectrometry (MS). Besides compounds from the environmental air, breath contains compounds from the upper airways as well as systemic metabolites, which are transported to the lungs after passing the blood-air barrier. In contrast to conventional technologies in metabolomics, a direct analysis of breath by secondary electrospray ionization coupled to high-resolution mass spectrometry (SESI-HRMS) allows for the simultaneous detection of a wide range of compounds. This showcases the methods capability for metabolic profiling and biomarker discovery, which has been demonstrated in multiple studies so far. However, the validation of potential biomarkers and of direct breath analysis for clinical applications has yet to be achieved.
In this thesis, several approaches were pursued to drive advances in SESI-HRMS breath analysis towards clinical application. Breath signatures were identified using either a SESI source coupled to a hybrid time-of-flight mass spectrometer (TripleTOF) or to a hybrid orbitrap mass spectrometer (QExactive). The high mass resolution of both analyzers, combined with their fast detection rate, enabled real-time tandem mass spectrometry (MS2 ) measurements in exhaled breath. This was necessary in order to attain metabolic profiling and it revealed metabolic insights into an asthma-specific breath signature. In this context, a strategy for compound identification was adjusted to enhance the confidence level of identified compounds. It included the identification via matching of fragment spectra from breath with human databases combined and then combined with additional pathway analysis. By the controlled introduction of various gas-phase compounds, it was possible to determine technical variations on SESI-HRMS instruments.
The diagnostic potential of SESI-HRMS based breath analysis was demonstrated on breath signatures found in patients with obstructive sleep apnea (OSA). In this work, the validation of OSA-associated markers, that were initially reported in a randomized-controlled study and validated in a follow-up study with larger sample size, was achieved. Confirming these markers in a more diverse study cohort in combination with instrumental changes, demonstrates the robustness of the validated markers and paves the way for future biomarker identification.
The applicability of breath analysis by SESI-HRMS was successfully extended to schoolaged children (age of 5-18 years). Untargeted breath analysis resulted in a distinct breath signature that distinguishes allergic asthmatics from healthy children. Following the compound identification on the most discriminative features, possible links to pathways involved in the pathophysiology of asthma were discovered. Given the non-invasive nature of SESI-HRMS sampling together with its metabolic profiling capability, the method showed its potential to significantly improve asthma phenotyping and early diagnosis of asthma even in preschoolaged children.
Moreover, a system was developed that mimics the conditions of exhaled breath in direct analysis. The system included a humidification and heating of gas-phase standards, as well as a serial dilution of gas-phase concentrations to parts per billion levels. The evaluation by single- and multi-component standard materials allowed for a much-needed inter-laboratory comparison and method validation. The daily use of the delivery system for standard measurements furthermore enabled a reliable monitoring of technical variations on SESI-HRMS. Additionally, this highlighted the need for implementing standardization procedures in direct breath analysis, such as routine measurements of quality control samples.
SESI sources are mainly used on two different mass analyzer technologies: QTOF and Orbitrap instruments. The source, however, is likely to be coupled to different instruments in the future as analyzer technologies evolve. In this work, a SESI-QTOF instrument and a SESI-Orbitrap setup were directly connected to each other to investigate the comparability of simultaneously analyzed breath profiles. These profiles revealed differences in generated adduct ions and the possibility of temperature-dependent fragmentation. Identifying the limit of comparability will generate a deeper understanding of inter-laboratory discrepancies, which will be especially important in multi-center studies going forward.
In conclusion, this work equally emphasizes the diagnostic value of breath analysis and addresses the necessary steps for its transition into the clinics. The applicability in young children, as well as the successful validation of breath markers, attest the technologys robustness. Implemented standardization strategies in direct breath analysis will eventually increase its impact for clinical use and for metabolomics.
