## 4 Conclusions  
This study presents a comprehensive analysis of the volatilome of _Theobroma grandiforum_ at three different maturation stages (medium, ripe, and overripe) using HS-SPME-GC-MS analysis and molecular networks. Additionally, the use of DoE for optimizing sample preparation and SPME conditions helped develop a simple extraction process, improving the sensitivity of VOC analysis. The conditions for the extraction of VOCs were achieved using 750 mg of the sample grounded with liquid nitrogen and an SPME fiber pre-equilibration time of 6 min. In addition, by DoE, the optimal exposition time and temperature were determined to be 27 min and 40 \({}^{\circ}\)C, respectively. The 96 VOCs exhibited variable abundances throughout the different maturation stages. The volatile profiles were analyzed using multivariate analyses, such as PCA and PLS-DA, which enabled the identification of specific VOCs that could serve as potential biomarkers for monitoring the ripening process of copoazu. Notable VOCs include \(\alpha\)-terpineol, _trans_-4-methoxythujane,linalo, 2-methylbutyl butanoate, 3-methylbut-2-enoic acid, 2-methylpentyl ester, unknown, vinyl ethanoate, and 2-methylpropyl hexanoate. Additionally, the ripe stage has emerged as a transitional stage in VOC production, with a potential biomarker showing a decrease in abundance in the overripe stage and absence in the medium stage due to the presence of precursors. Compounds that differentiated the odor of copoazu at different stages of maturation were identified. In the mature stage, the fruity, sweet, citric, and floral odors are attributed to esters and terpenes, whereas in the overripe stage, the alcohols are related to the herbal, woody, and turpentine odors. On the other hand, the molecular network analysis enabled the visualization of structural relationships between various groups of volatile compounds, including esters, alcohols, monoterpenoids, and carbonyl compounds. The molecular network of VOCs evidenced the chemical similarities observed in the different families of compounds that correlated due to their biosynthetic pathways. The findings of this study provide a solid foundation for future studies. Further exploration of the biosynthetic mechanisms underlying VOCs production and the application of biomarker monitoring to optimize copoazu harvesting and processing are possible prospective applications in the food industry. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/molecules30061209/s1](https://www.mdpi.com/article/10.3390/molecules30061209/s1)]([https://www.mdpi.com/article/10.3390/molecules30061209/s1](https://www.mdpi.com/article/10.3390/molecules30061209/s1)). Figure S1: Influence of sample treatment on the total area of 6 majority VOCs. (a) Total area and (b) area of each compound. The results are expressed as the mean \(\pm\) SEM of duplicate analyses. Different letters in each composite indicate a significant difference at \(p\leq 0.05\), as determined by the LSD-Fisher test. Figure S2: Optimization of the sample amount. Results were expressed as the mean \(\pm\) SEM of duplicate analysis. Different letters in each compound indicate a significant difference at \(p\leq 0.05\), as determined by the LSD-Fisher test. Figure S3: (a) Pareto plot of standardized effects for 3-methylbutyl alcohol and (b) area response surface of isoamyl alcohol over time vs. extraction temperature. Figure S4: (a) Pareto plot of standardized effects for hexanal and (b) hexanal area response surface for time vs. extraction temperature. Figure S5: (a) Pareto plot of standardized effects for ethyl 2-methylbutanoate and (b) area response surface of ethyl 2-methylbutanoate over time vs. extraction temperature. Figure S6: (a) Pareto plot of standardized effects for 3-methylbutyl ethanoate and (b) area response surface of isoamyl acetate for time versus extraction temperature. Figure S7: FTIR spectra of ripening stages of copoazu. Medium-ripening stage (black), ripe (red), and very ripe (green).