## 2 Results and Discussion
### Shelf-Life Study
#### 2.3.1 Evolution of Chemical Characteristics over Time
The increase in K\({}_{270}\) is associated with the formation of conjugated trienes and secondary oxidation products such as dialdehydes. Therefore, unlike peroxide value and K\({}_{232}\), the larger bottle did not exhibit a clearly beneficial effect on K\({}_{270}\). **Phenolic compounds**: Figure 2 shows the evolution of the total phenolic content (TPC), the main individual phenols (i.e., hydroxytrosol, tyrosol, oleacein, oleocanthal), and the hydrolysis ratio (i.e., the ratio of the sum of tyrosol and hydroxytrosol divided by TPC) over time, while Table 7 summarizes the significance of the effect of each factor and their interaction, as evaluated using a 3-way ANOVA. Additional data are provided in Table S5 of the Supplementary Material. Phenolic compounds are among the most important molecules characterizing high-quality extra virgin olive oils (EVOOs) and, along with VOCs, represent a key parameter for distinguishing the quality of different EVOOs. These compounds confer multiple attributes to EVOO, including antioxidant properties that enhance shelf-life, sensory properties (contributing to the oil's typical bitter and pungent characteristics), and nutritional properties that help reduce the risk of various degenerative diseases [21; 49]. The most distinctive EVOO phenols are secioridoid derivatives of oleuropein and ligstroside. This report focuses on phenolic molecules relevant for profiling of Montespertoli's EVOOs: (i) total phenolic content (TPC) that provides a general indication of how rich the EVOO is in phenolic compounds; (ii) oleacein and oleocanthal, often the most abundant phenols in high-quality EVOO, though their content vary depending on cultivar; (iii) tyrosol and hydroxytrosol, as final degradation products of the EVOO's phenolic compounds resulting from hydrolytic, esterase, and intramolecular rearrangement processes. In freshly produced high-quality EVOO, their concentration is low and negligible relative to the TPC, but they increase over time, and high levels indicate a degraded phenolic profile; (iv) hydrolysis ratio, defined as the ratio between the sum of tyrosol and hydroxytyrosol to the total phenolic content. The higher this value, the more advanced the hydrolytic degradation of the phenolic profile, indicating a greater degree of EVOO aging [39; 50; 51]. Tyrosol and Hydroxytyrosol: Table 7 shows that the concentrations of these two molecules varied significantly with storage time, sample type, and their interaction. No significant effects were observed due to bottle size. Hydroxytyrosol values ranged from an initial 3.6 mg/kg to 13.5 mg/kg for the 250 mL bottle and 13.2 mg/kg for the 500 mL bottle in EVOO 1, and from 3.6 mg/kg to 6.4 mg/kg (250 mL) and 6.6 mg/kg (500 mL) in EVOO 2. Tyrosine values ranged from an initial 3.5 mg/kg to 4.8 mg/kg (250 mL) and 4.9 mg/kg (500 mL) in EVOO 1, and from 5.9 mg/kg to 5.6 mg/kg (250 mL) and 5.5 mg/kg (500 mL) in EVOO 2. These values are also acceptable for high-quality oils after one year; only in the case of hydroxytyrosol in EVOO 1, a significant increase occurred at t3, suggesting the  
Figure 2: Evolution of phenolic compounds (**A**) hydroxytyrosol, (**B**) tyrosol, (**C**) oleacein, (**D**) oleacein, (**E**) hydrolysis ratio, (**F**) total phenolic content) in the two oils stored in the two bottles over storage. t0, time 0; t1, 2 months; t2, 5 months; t3, 12 months; 250 mL and 500 mL are the bottles’ sizes. In each graph, different letters over the bars indicate significant differences at \(p<0.05\). effect of each factor and of their combination as evaluated using 3-way ANOVA. Detailed data are also provided in Table S6 of the Supplementary Material. Tocopherols are lipophilic antioxidant molecules present in oils as \(\alpha\), \(\beta\), \(\gamma\), and \(\delta\) isoforms [8], with the \(\alpha\) form being predominant in EVOO [53]. In this study, \(\beta\)- and \(\gamma\)-forms are reported together, as they are not separated chromatographically using the method applied.