Shareable abstract Exhaled breath analysis, utilising VOCs, offers promise in identifying SSc-ILD patients, aiding targeted treatment. Further multicentric studies are crucial for validation and exploring longitudinal VOC changes for comprehensive disease management.https://bit.ly/4aKMYif

Exhaled breath analysis represents a promising non-invasive approach for disease monitoring through volatile organic compounds (VOC) detection. However, the lack of standardized sam-pling methods do not enable direct clinical translation. This study compared three widely used offline breath sampling techniques (Tedlar® bags, BioVOC-2®, and ReCIVA®) using the estab-lished peppermint benchmarking protocol and comprehensive two-dimensional gas chroma-tography coupled to mass spectrometry (GC×GC-MS). Seven healthy participants completed the peppermint experiment, with breath samples col-lected at multiple time points following capsule ingestion. Washout curves for targeted terpe-noid compounds were analyzed to assess analytical performance, reproducibility, and back-ground contamination across devices. Clinical feasibility was evaluated through focus groups with clinicians, researchers, and study participants. Tedlar® bags demonstrated reliable performance with lowest overall pooled relative standard deviations, though sensitive to exogenous contamination. ReCIVA® showed higher overall vari-ability, superior selectivity and reduced background interference compared to Tedlar® bags (p<0.01). However, ReCIVA® showed higher complexity, cost, reduced comfort and potential for saliva contamination during extended sampling. BioVOC-2® offered operational simplicity but was limited by small sampling volume (129 mL) reducing its sensitivity and manual handling variability. No single device emerged as universally optimal. Tedlar® bags, when accompanied by rigorous standard operating procedures (SOPS), remain most suitable for large-scale studies, BioVOC-2® for rapid targeted screening, and ReCIVA® for controlled research requiring high selectivity. Successful clinical implementation will require balancing analytical performance with practical considerations including patient comfort, cost-effectiveness, and workflow integration. These findings support ongoing standardization efforts within breathomics community and extend peppermint database for exhaled breath sampling.

Systemic sclerosis (SSc) is an autoimmune disease causing inflammation, blood vessel damage, and collagen deposition. Interstitial lung disease (ILD) is common in SSc, leading to significant mortality. Early detection of SSc-ILD is crucial, but current biomarkers are limited. Our previous research identified distinct volatile organic compound (VOC) patterns in SSc patients' breath. This study aims to establish standardized breath analysis procedures, and assess VOCs' potential in predicting SSc-ILD. Two expert medical centers, the University Hospital of Liège (CHU), Belgium, and Maastricht University Medical Center (MUMC+), the Netherlands, collaborated in studying and recruiting 21 SSc patients and 21 SSc-ILD patients. Nine VOCs were identified as discriminatory between SSc and SSc-ILD, outperforming traditional blood markers. The statistical model based on these markers achieved an AUC of 0.82, accuracy of 85%, sensitivity of 77% and a specificity of 100% for indentifying ILD phenotype, comparable to traditional lung function tests. A correlation was also observed between the functional respiratory parameters (i.e., DLco and FVC% predicted value) and the VOCs. Additionally, our study confirmed the potential of four terpenes, observed in both studies, in distinguishing SSc patients. Methodological SOPs for multi-center studies were developed and validated. This study demonstrates the potential of breath analysis and in particular markers discovery in understanding SSc metabolic changes and could aid prompt ILD treatment. Future research will explore VOC changes in early-stage SSc-ILD and treatment response. This study paves the way for improved diagnosis and management of SSc-ILD.