Novel Impingement-Plate Mouthpieces: Enhancing Data Quality in Breathomics Analysis via Direct High-Resolution Mass...

Breath sampling mouthpieces are essential for breathomics research, as they effectively remove saliva contamination during sampling. However, for real-time online mass spectrometric breath analysis, the widely adopted filter-based mouthpieces substantially compromise data quality due to analyte adsorption and background interference. In this study, guided by the inertial impaction principle, we designed and fabricated three impingement-plate mouthpiece types via 3D printing using biocompatible, low-porosity poly(lactic-co-glycolic acid). Computational fluid dynamics simulations and salivary amylase validation experiments confirmed efficient saliva droplet interception by the proposed configurations. Targeted detection of a ten-component gas standard mixture by secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) demonstrated that impingement-plate types exhibited far fewer statistically significant deviations from the control group compared with the filter-based mouthpiece. Among the three designs, the reverse-raked configuration yielded responses closest to the control for most analytes. The types also provided improved signal stability and reproducibility, with interunit coefficients of variation (CVs, n = 10) of 6.43-29.84%, compared with 28.91-66.36% for the filter-based mouthpiece. Principal component analysis based on high-purity nitrogen purging tests showed that the impingement-plate mouthpiece groups clustered closer to the control group than the filter group, suggesting minimal background interference. Untargeted breath profiling confirmed that mass spectral features acquired with impingement-plate mouthpieces were highly consistent with those obtained from filter-free sampling, while the filter-based mouthpiece induced deviations in breath metabolic signatures. Overall, inertial impaction-based designs mitigate the trade-off between saliva droplet removal and analyte transmission, reducing mouthpiece-derived bias and improving data fidelity and analytical robustness in real-time breathomics.