## 4 Discussion  
The aim of the study was to identify potential metabolites that can differentiate between patients with different stages of COPD. We collected exhaled air samples from patients within the same age range, including both patients with exacerbations and patients without exacerbations. We successfully employed an analytical methodology that involved using RTubeVOC for air sample collection and solid phase microextraction (PDMS/CAR/DVB-30/50 um) for volatile compound extraction. Gas chromatography coupled with mass spectrometry was used for the analysis of the extracted compounds. To analyze the complex data obtained from the analysis, multivariate statistical analysis was performed. As a result, four compounds were identified: Heptane,2,2,4,6,6-pentamethyl, gamma-terpinene, 2-ethylhexanol, and Undecane. It is worth noting that 2-ethylhexanol belongs to the alcohol compound class, while the other three compounds belong to the hydrocarbon class. The identification of these compounds was done with a confidence level greater than 95%.  
The study faced great challenges in collecting air samples from patients due to the coronavirus situation in Greece. With a larger number of samples, the results could potentially be more representative of the disease and provide a better understanding of COPD. It's important to note that there are currently no studies available that specifically compare the two stages of COPD (patients with exacerbations and patients without exacerbations). Existing literature mainly focuses on comparisons between COPD patients and healthy subjects, lung cancer patients, or asthma patients [17].  
Nevertheless, this study contributes valuable evidence by demonstrating differences in the breath profiles of patients with COPD and exacerbations compared to patients with COPD without exacerbations. Four VOCs were identified in the analysis. It was found through literature research that undecane and n-terpinene have been reported as identified biomarkers in online COPD databases [17; 18]. The remaining two biomarkers are likely among the many yet to be certified biomarkers, as there are over 3000 metabolites in exhaled air, and approximately 500 have been identified so far. Our suggested approach addresses the primary limitation in the analysis of exhaled-breath condensate, which is likely linked to the challenge of standardizing sample collection. With respect to electronic nose, besides its portability, it is not equipped to offer structural elucidation for distinguishing VOCs within different groups. However, our method cannot be compared to the real-time techniques that provide minimized sample handling with low associated risk of sample contamination.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/chemosensors11100542/s1](https://www.mdpi.com/article/10.3390/chemosensors11100542/s1)]([https://www.mdpi.com/article/10.3390/chemosensors11100542/s1](https://www.mdpi.com/article/10.3390/chemosensors11100542/s1)), Figure S1: A typical SPME/GC-MS chromatogram of an exhaled breath sample of patient (A) with exacerbations and (B) without exacerbations.  
Conceptualization: C.C., V.S. and K.K.; methodology, L.L., C.C., V.S. and K.K.; software, L.L. and K.E.; validation, L.L., C.C. and V.S.; formal analysis, L.L.; investigation, L.L., C.C. and K.E.; resources, K.K. and V.S.; data curation, L.L., K.E. and V.S.; writing--original draft preparation, L.L., C.C. and K.E.; writing--review and editing, L.L., C.C., K.E., K.K. and V.S.; visualization, L.L. and K.E.; supervision, K.K. and V.S.; project administration, V.S.; All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** Not applicable.  
**Conflicts of Interest:** The authors declare no conflict of interest.