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 degrees C-40 degrees 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 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.
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