Challenges in the identification and quantitation in on-line breath analysis

The identification and quantitation of volatile organic compounds (VOCs) in exhaled human breath has attracted considerable interest due to its potential application in medical diagnostics, environmental exposure assessment, and forensic applications. Secondary electrospray ionization-mass spectrometry (SESI-MS) is a method capable of detecting thousands of VOCs. Nevertheless, most studies using SESI-MS for breath analysis have relied primarily on MS¹measurements for identifications and quantification, which are susceptible to misassignments and errors. In this study, we targeted several endogenous compounds (C5 to C10 aldehydes, limonene and pyridine), known to occur in breath. These compounds were measured and quantified in exhaled breath from 12 volunteers over several days using three different acquisition methods: full scan, targeted selected ion monitoring and parallel reaction monitoring. These methods were used for identification and quantification by comparing with measurements of external standards. High-abundance features such as limonene and pyridine were successfully identified and quantified in exhaled human breath with all three methods, with MS²measurements supporting identification, albeit with limitations to separate between limonene andα-/β-pinene. For low-abundance features, the study highlights the challenges of false assignments in SESI-MS, even with MS²measurements. This was demonstrated in the case of aldehydes, which could not be reliably separated from isomeric ketones present in breath, leading to incorrect quantification.