Amino acid profiles in exhaled breath condensate: evaluation of collection methods and diurnal variation Using...

Exhaled breath condensate (EBC) is a non-invasive biological fluid that reflects metabolic activity in the respiratory tract. It has attracted interest as a potential source of biomarkers for respiratory diseases. EBC contains amino acids that play central roles in metabolism and disease pathophysiology; these amino acids may therefore serve as promising biomarkers. However, the analysis of these amino acids remains limited because of their low concentrations, and their diurnal variation remains unclear. Furthermore, variability in collection devices and the risk of saliva contamination can make the reliable analysis of amino acids in EBC difficult. In this study, we developed a sensitive and robust method for analyzing amino acids in EBC using hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry. Adding succinic acid to the mobile phase increased ionization efficiency. In post-column mixing experiments, addition of succinic acid improved peak areas, with more than three-fold increases observed for 10 of 16 amino acids. We conducted preliminary human studies to evaluate the effects of collection devices, saliva contamination, and diurnal variation. Both syringe-based and commercial RTube devices enabled EBC collection in a small sample size (n = 5); however, the RTube yielded larger sample volumes. Despite differences in EBC yield, amino-acid concentrations showed largely similar trends between the two devices in most participants, although sample-specific variation was observed. Diurnal variation in healthy subjects (n = 11) revealed peak amino-acid levels after overnight fasting, followed by a secondary rise after lunch. Differences in amino-acid composition ratios between individuals were less pronounced than differences in absolute concentrations. This study demonstrates a practical method for quantifying amino acids in EBC and highlights the impact of technical and biological factors on measured levels. These preliminary findings support further evaluation of EBC amino-acid profiles as potential biomarkers for respiratory diseases.