The cannabinoid content within the high THCA chemovar was affected by the hexanoic acid treatment (Figure 1). On the day of harvest (t₀), the concentrations of CBGA, CBDA, and total minor cannabinoids (cannabinoids with concentration below 1 DW%) were significantly higher by 16–22% in the inflorescences treated with hexanoic acid (Figure 1). However, at d6, following the drying and curing procedures, only the CBGA concentration was significantly elevated by 14% in the hexanoic acid treatment group in comparison to the reference group (Figure 1). The higher CBGA concentration at t₀ and d6 in the hexanoic acid treatment group supports the hypothesis that pre-harvest hexanoic acid treatment on cannabis inflorescence can enhance the formation of major cannabinoids such as CBGA. This observation also provides an explanation for the higher cannabinoid concentrations observed in the hexanoic acid treatment group, as CBGA serves as the primary metabolite to form THCA, CBDA, and CBCA [6,7].

Furthermore, no statistically significant alterations were detected in the concentrations of the majority of the cannabinoids examined throughout the drying and curing processes, with the exception of THC concentration in the reference group, which increased by 47%, and CBG concentration in the hexanoic acid group, which decreased by 27% (Figure 2, Table S1). In both cases, the absolute concentration changes were less than 0.08 DW% (Table S1).

At the end of the storage period (d130), a noticeable difference in cannabinoid levels was found among the different groups studied (Figure 1). The highest level of the main cannabinoid, THCA, on d130 was found in the Hex8mM-VAC group, followed by REF-VAC, Hex8mM-air, and REF-air (Figure 1a). Hex8mM-VAC emerged as the only treatment group wherein the THCA concentration was significantly higher (by 38%) compared to the reference group stored in polyethylene plastic sealed packages (REF-air, Figure 1a). These findings imply that for the preservation of elevated THCA concentrations over an extended duration, the fine-tuning of both pre- and post-harvest processes is required. Additionally, the Hex8mM-VAC group exhibited the highest concentration of CBGA, CBDA, and CBCA, after four months of storage (22–45% higher than REF-air, Figure 1b–d). No significant differences in the concentration of the remaining minor cannabinoids among the different groups were observed (Figure 1e–h). Consequently, the highest total cannabinoid, total minor cannabinoid, and total THC content at d130 were observed in the Hex8mM-VAC group (Figure 1k,l).

Following four months of storage (d130), the concentration of THCA, acting as the “reservoir” for THC, decreased in the various groups examined by 4–23%, with the minimal and statistically non-significant reduction being recorded in the Hex8mM-VAC group, in comparison to the conclusion of the drying and curing phase (d6, Figure 2a). On the other hand, the biggest decrease was observed in the REF-air group, highlighting the need for proper storage and pre-harvest treatment (Figure 2a). This latter observation aligns with the results documented in prior studies, where a 15–20% decrease in THCA concentration was noted post-storage under similar conditions [20,21], as well as with a recent study revealing 15–30% lower THCA concentrations in dried cannabis inflorescence as compared to the levels indicated on dispensed package labels [24]. Consequently, a revision of the prevailing industrial storage conditions is warranted. Surprisingly, the reduction in THCA levels does not result in a corresponding increase in THC or in any other hereby analyzed degradation products of THCA [6]. The sole THCA degradation product identified, CBNA, yielded a negligible concentration of approximately 0.02 DW% after four months of storage (Figure 1i). The absence of mass balance concerning THCA decline was similarly noted in other studies [19,20,21]. However, in stark contrast to previous findings [6,12,19,25], the decarboxylation of acidic cannabinoids to neutral cannabinoids was not observed in the current study during the four-month storage period. On the contrary, the concentrations of neutral cannabinoids, THC and CBG, significantly declined by 22–78% during storage (Figure 2g,h). Due to the concentration decrease in both THCA and THC during storage, the total THC content also decreased in the REF-air and REF-VAC groups (Figure 2l). Apart from CBCA and (-)-Δ9-trans-tetrahydrocannabiorcolic-C4 acid (THCA-C4), which sustained a stable concentration throughout storage in all groups studied, the remaining cannabinoids displayed a significant concentration decline during storage, with the most pronounced decrease noted in the REF-air group and the slightest decrease observed in the Hex8mM-VAC group (Figure 2). Taken altogether, the treatment group that yielded the highest cannabinoid concentration after four months of storage was the Hex8mM-VAC group.

Given that cannabis-based products are classified as medicinal products, they are subjected to strict medical grade regulations [26]. According to the EU guidelines for the shelf life estimation of drug substances, the shelf life of a medical product refers to the time period during which the product retains 95% of its major cannabinoids’ content determined at the beginning of the storage [17]. Consequently, solely the inflorescences from the Hex8mM-VAC treatment group, which exhibited a 96% relative THCA concentration at the end of the storage period, are considered unexpired, indicating that they remain suitable for the intended use for a minimum of four months (Figure 2a). Furthermore, the THCA concentration in the Hex8mM-VAC treatment group at the end of the storage exceeded the initial concentration of the reference groups at d6 by 6%. All other treatment groups exhibited a 77–90% relative THCA concentration at the end of the storage period and are considered expired (Figure 2a). These findings indicate that optimal pre-harvest conditions coupled with optimal post-harvest storage increased the product’s shelf life and consequently the medicinal efficacy of the final product.

Recent studies investigating the influence of integrated parameters on cannabinoids’ stability during storage mainly focused on post-harvest parameters—e.g., the combination of temperature and light exposure [19,20,21,22,27]—while our study is the first to examine the specific integrated effect of both pre- and post-harvest approaches, which have not been considered so far. Moreover, the hexanoic acid spray results obtained in this study are in line with the impact of hexanoic acid on the secondary metabolite composition in other plant species studied such as tomato and citrus [13,15].

The influence of hexanoic acid treatment on the mono-terpene content within the high THCA chemovar was less pronounced than in the case of cannabinoids (Figure 3). At harvest day (t₀), the concentrations of all mono-terpenes were similar and not statistically different in both reference and hexanoic acid treatment groups (Figure 3). Moreover, no differences in the concentrations of these mono-terpenes were observed between the reference and the hexanoic acid treatment groups after the drying and curing processes (d6, Figure 3), except for α-terpinenol which displayed a significantly higher concentration by 25% in the hexanoic acid treatment group than the reference group (Figure 3h).

At the end of the drying and curing processes (d6), the concentration of d-limonene and β-myrcene decreased by 21–48% in all of the treatment groups as compared to t₀, with the reference group displaying lower concentration values than the hexanoic acid treatment group (Figure 4a,d, Table S1). No significant change in α-pinene concentrations was observed between t₀ and the end of the drying and curing processes (d6, Figure 4b). On the other hand, at d6, the concentrations of (-)-β-pinene, linalool, fenchol, pinalol, and α-terpinenol decreased significantly by 13–34% only in the reference group (Figure 4c,e–h, Table S1), indicating that the hexanoic acid pre-harvest treatment maintained steady-state levels of mono-terpenes during the entire drying and curing period.

At the end of the storage period (d130), substantial differences in the mono-terpene content between the different groups were found (Figure 3). Except β-myrcene, the highest mono-terpene concentrations at d130 were observed in the Hex8mM-VAC group, followed by Hex8mM-air, REF-VAC, and REF-air groups (Figure 3). The concentrations of the various mono-terpenes in the Hex8mM-VAC group at d130 were higher by 20–82% as compared to the REF-air group (Figure 3). Moreover, both hexanoic acid pre-harvest treatment groups (Hex8mM-VAC and Hex8mM-air) exhibited significantly higher concentrations of the mono-terpenes d-limonene, (-)-β-pinene, linalool, and fenchol as compared to both reference groups stored under vacuum and air conditions (REF-VAC and REF-air, Figure 3a,c,e,f), indicating that hexanoic acid treatment maintained steady-state levels of the aforementioned aroma compounds during the storage period. The preservation of major aroma components is of the utmost importance, due to their contribution to the overall pharmacological activity of cannabis products as well as the appealing aroma of the inflorescence, specifically sought by both patients and recreational users.

After four months of storage, the concentrations of α-pinene, (-)-β-pinene, and pinalol remained stable across all examined groups, with the exception of (-)-β-pinene in the REF-air group and pinalol in the Hex8mM-air group, where a statistically significant decrease of 13–14% was observed (Figure 4b,c,g). On the other hand, the concentrations of β-myrcene, fenchol, and α-terpineol significantly decreased in all of treatment groups by 19–51%, with the lowest reduction observed in the Hex8mM-VAC group (Figure 4d,f,h). The Hex8mM-VAC group was the only treatment group in which the concentration reduction of the major mono-terpene, d-limonene, was not statistically significant as compared to d6 (Figure 4a). Taken altogether, the integrated effect of the pre-harvest hexanoic acid treatment followed by vacuum storage under optimal temperature and humidity conditions yielded stable major cannabinoid and mono-terpene concentrations, thereby substantially increasing the products’ shelf life and therefore maintaining the quality and efficacy of the cannabis inflorescence.

The hexanoic acid treatment affected the sesqui-terpene content differently than the mono-terpene content (Figure 5). At harvest day (t₀), the concentrations of β-caryophyllene, α-humulene, (-)-α-bisabolol, γ-elemene, and α-selinene were significantly higher in the reference group as compared to the hexanoic acid treatment group (Figure 5a,b,d,e,i). Nevertheless, these differences were not maintained at the end of the drying and curing period (d6) due to a significant decrease of 13–21% in their concentrations, observed solely in the reference group (Figure 5a,b,d,e,i, Figure 6a,b,d,e,i). Moreover, at d6, β-eudesmene displayed a significant concentration decline of 21% as compared to t₀ only in the reference group (Figure 6f, Table S1). These results support the observation that the hexanoic acid pre-harvest treatment maintained steady-state levels of terpenes during the entire drying and curing period.

At the end of the storage period (d130), the concentration differences of several sesqui-terpenes between the Hex8mM-VAC group and the REF-air group mirrored those observed for both mono-terpenes and cannabinoids (Figure 5). The concentration of the sesqui-terpenes, β-caryophyllene, (-)-guaiol, γ-eudesmol, β-eudesmol, and α-selinene were significantly larger only in the Hex8mM-VAC group as compared to the REF-air group by 19–43% (Figure 5a,c,g–i). On the other hand, no significant differences were observed in the concentration of α-humulene, (-)-α-bisabolol, γ-elemene, and β-eudesmene after four months of storage between the different groups (Figure 5).

At d130, the concentration of the sesqui-terpenes, α-humulene, (-)-guaiol, γ-elemene, β-eudesmene, and γ-eudesmol remained stable and not statistically significant as compared to d6 in all treatment groups studied (Figure 6). On the other hand, β-caryophyllene and β-eudesmol displayed a significant concentration decline (by 15–22%) only under non-vacuum storage conditions, (-)-α-bisabolol displayed a significant concentration decline (by 19–22%) in all groups examined except REF-air, while α-selinene exhibited a significant concentration decrease in all treatment groups (by 23–43%, Figure 6). Taken altogether, these results suggest that sesqui-terpenes are less susceptible to degradation than mono-terpenes during long storage times. Moreover, these results suggest that sesqui-terpenes are less influenced by the integrated effect of pre-harvest hexanoic acid treatment and vacuum storage.

Significant differences in total terpene content between the treatment groups were found only after four months of storage. The highest mono-terpene content was observed in the Hex8mM-VAC group, followed by the Hex8mM-air group, the REF-VAC group, and the REF-air group (Figure 7a). This indicates that hexanoic acid treatment had a greater impact on the mono-terpene content preservation than storage under vacuum without pre-harvest spraying. During drying and curing, the mono-terpene content in all groups examined, except the Hex8mM-VAC group, resulted in an 18–26% significant decrease (Figure 8a). On the other hand, no significant differences in sesqui-terpene content were observed between the different groups under all time points investigated, possibly since they are less susceptible to degradation and are less volatile than the mono-terpenes (Figure 7b) [28]. The highest total terpene content after four months of storage was observed in the Hex8mM-VAC group, which was the only treatment group that was significantly different from the REF-air group (Figure 7c).

The literature reports about the impact of various post-harvest conditions on terpenes’ preservation in cannabis inflorescence are conflicting [19,22]. Grafström et al. found that the concentration of numerous sesqui-terpenes remained stable during four months of airtight storage conditions, in agreement with our study outcome [22]. On the other hand, Milay et al. found that the mono- and sesqui-terpene content significantly decreased after four months of storage regardless of the storage temperature [19].

The application of hexanoic acid to citrus trees resulted in an enhanced content of aromatic volatile compounds [13]. However, in the current study, this effect was only discerned in the cannabis inflorescence at the conclusion of the storage period and not during the drying and curing stages. Additionally, it was observed that the reference group underwent a significant reduction in 11 distinct aroma compounds during drying and curing, whereas the hexanoic acid group did not exhibit such a decrease. These findings imply that hexanoic acid treatment in cannabis may aid in the preservation of volatile compounds during the initial post-harvest stages and throughout storage, rather than increasing their production as observed in citrus [13].

The combined application of pre-harvest hexanoic acid spraying and post-harvest vacuum storage has shown to either match or enhance cannabis inflorescence shelf life in terms of major cannabinoid retention [19,20,21]. Trofin et al. observed a 17% reduction in THCA concentration after five months of storage at 4 °C, whereas Reason et al. noted a 10% reduction in THCA concentration after two months of storage at 6 °C [20,21]. Compared to these findings, our Hex8mM-VAC treatment exhibited a reduced THCA degradation, with only a 4% decrease in relative concentration after four months.

Milay et al. documented a 15% drop in THCA relative concentration over 12 months of storage at 4 °C, translating to a 5% decrease over a four-month period, slightly more than our 4% decrease following Hex8mM-VAC treatment [19]. Furthermore, a significant reduction in terpene levels during four months of storage at 4 °C has been reported in Milay et al. [19]. Milay et al. found a 39–64% reduction in the concentrations of α-pinene, (-)-β-pinene, β-myrcene, linalool, fenchol, d-limonene, α-terpinenol, β-caryophyllene, α-humulene, and (-)-α-bisabolol after four months of storage at 4 °C [19]. In contrast, our Hex8mM-VAC method resulted in a less pronounced decline in terpenes, with decreases of 20–23% for fenchol, α-terpinenol, and (-)-α-bisabolol; a 37% decrease for β-myrcene; while the levels of α-pinene, (-)-β-pinene, linalool, d-limonene, β-caryophyllene, and α-humulene remained constant throughout the storage period. Thus, the integrated effect of hexanoic acid spraying and vacuum storage appears to surpass the performance of storage conditions reported in the literature.