Real-time breath analysis towards a healthy human breath profile.

The direct analysis of molecules contained within human breath has had significant implications for clinical and diagnostic applications in recent decades. However, attempts to compare one study to another or to reproduce previous work are hampered by: variability between sampling methodologies, human phenotypic variability, complex interactions between compounds within breath, and confounding signals from comorbidities. Towards this end, we have endeavored to create an averaged healthy human 'profile' against which follow-on studies might be compared. Through the use of direct secondary electrospray ionization combined with a high-resolution mass spectrometry and in-house bioinformatics pipeline, we seek to curate an average healthy human profile for breath and use this model to distinguish differences inter- and intra-day for human volunteers. Breath samples were significantly different in PERMANOVA analysis and ANOSIM analysis based on Time of Day, Participant ID, Date of Sample, Sex of Participant, and Age of Participant (p< 0.001). Optimal binning analysis identify strong associations between specific features and variables. These include 227 breath features identified as unique identifiers for 28 of the 31 participants. Four signals were identified to be strongly associated with female participants and one with male participants. A total of 37 signals were identified to be strongly associated with the time-of-day samples were taken. Threshold indicator taxa analysis indicated a shift in significant breath features across the age gradient of participants with peak disruption of breath metabolites occurring at around age 32. Forty-eight features were identified after filtering from which a healthy human breath profile for all participants was created.