Chemical Analysis of Deep-Lung Fluid Derived from Exhaled Breath Particles

Breath particles generated deep within the lung provide noninvasive access to sampling nonvolatiles in peripheral airway lining fluid. However, background contamination, their variable production among subjects, together with a huge unknown dilution when using the common breath condensate method for collection has limited their use for quantitative biomarker analysis. Instead, we first capture and dry the particles in a flexible chamber followed by accurate optical particle characterization during their collection for chemical analysis. By decoupling breathing and aerosol sampling airflows, this sequential approach not only accommodates all types of breathing routines but also enables the use of a variety of aerosol samplers for downstream biomarker analysis. Using ²³Na NMR, we measured 0.66 M Na in dry particles collected on a filter, which suggests that dehydration reduces their volume by a factor of ∼ 5.5 based on known Na levels in lung fluid. ¹H NMR revealed 0.36 and 0.68 M phosphocholine lipids in dried particles collected from two volunteers, presumably enriched to these levels relative to literature values derived from bronchoalveolar lavage fluid due to the film-bursting mechanism that underlies breath particle generation. Decoupling of breath collection and aerosol capture enabled the design of an impactor sampler with 72% efficiency. This impactor minimizes reagent and handling-related contamination associated with traditional filters by collecting dry particles directly in a microreactor for subsequent derivatization and quantification by mass spectrometry. The method is demonstrated by quantifying subnanogram amounts of urea from breath particles, corresponding to lung fluid urea concentrations consistent with literature blood plasma values.