*3.2. GC-MS-O Qualitative Analysis: Identification of Flavoring Additives*

The sensory evaluation report by GC-MS-O analysis of e-liquids ID A 1–5 is shown in Table 3. Molecular formula, CAS number and retention time (TR), expressed in minutes, of identified odor-active compounds, as well as the intensity of the odor perceived and the associated qualitative description provided by both trained panelists, are reported. GC/MS-O analysis of the sample 1-A with labelled flavor Coca cola allowed to distinctly identify 4 odor-active compounds: ethoxyethane, 2-ethoxybutane, camphene, and γ-terpinene. In more detail, the integration of chromatographic data with sensory perception revealed that the first odorous stimulus perceived by both assessors with intensity 3 (clear odor) and qualitatively described with the descriptor 'sweet' was associated with ethoxyethane eluted at 2.8 min. The odor-active compounds 2-ethoxybutane and γ-terpinene, eluted at 4.8 and 18.1 min respectively, were associated with the characteristic flavor of coca cola beverage and related to the overall flavor perceived during the preliminary odor test with the refill liquid. More specifically, 2-ethoxybutane was perceived by both panelists with intensity 3 and described as coca cola-like flavor while γ-terpinene was perceived by both the panelists with intensity 2, described with the descriptor 'bitter' and referred to the bitter aftertaste of coca cola. Another odor-active compound detected at the olfactory port and chromatographically identified was camphene, perceived by both assessors with intensity 2 and associated with citrus and fresh notes. The odor-active compounds 2-ethoxybutane, camphene and γ-terpinene are all classified by FEMA as flavoring agents with a specific flavor profile. 2-ethoxybutane is associated with the flavor profile 'floral' while camphene and γ-terpinene to the flavor profile 'camphor/oil' and 'bitter/citrus' respectively. Other sources e.g., The Good Scents Company (TGSC) Information System reports a more detailed flavor profile of camphene including minty, fresh, woody and citrus notes depending on the concentration confirming, in part, the assessors' olfactory perception. Finally, as shown in Table 3, two odorous stimuli although distinctly perceived at the olfactory port approximately at 8.9 and 21.3 min were not identified due to chromatographic peaks not sufficiently intense to allow accurate identification. The lack of clear correspondence between sensory perception and chromatographic data highlights that, despite the potentialities of GC-MS-O technique, in certain cases the sensory perception of human nose is more sensitive than the analytical detection as reported by Plutowska et al., 2008 [40]. The GC-MS-O analysis of e-liquid 2-A with the characteristic kiwi flavor resulted in the identification of seven odor-active compounds. Most of the odorous stimuli were qualitatively described by assessors with the odor descriptors 'sweet' and 'fruity'. The odor-active compounds identified, in order of chromatographic elution, were: ethoxy ethane (sweet, 2.8 min), ethyl acetate (aromatic/alcoholic, 3.6 min), 2-ethoxybutane (sweet/fruity, 4.8 min), methyl butanoate (fruity, 5.3 min), ethyl butanoate (fruity, 7.6 min), ethyl 2-methyl butanoate (fruity, 9.3 min), and methyl hexanoate (fruity, 12.3 min). Two were in common with e-liquid 1-A, i.e., ethoxy ethane and 2-ethoxy butane perceived by both assessors with intensity 3 and 2, respectively. The esters methyl butanoate, ethyl butanoate, ethyl 2-methylbutanoate, and methyl hexanoate are odor-active compounds with fruity attributes and represent a characteristic portion of the volatile aroma profile of fruits. They are also classified by FEMA as flavoring agents and are primarily used to impart fruity flavor in foods and beverages. Ethyl acetate is also included in the FEMA list of flavoring agents (with specification as food additive, carrier solvent) but its flavor profile is based on aromatic, brandy, and grape odor notes. Among the 'sweet' and 'fruity' odorous stimuli, both the assessors clearly indicated the one associated with the characteristic kiwi flavor, with odor intensity equal to 3. Comparing GC-MS results with the sensory response provided by both the panelists, ethyl 2-methyl butanoate was identified as the odor-active compound responsible of the kiwi flavor of the refill. This specific ester has been already identified in previous investigations by GC-MS and GC-MS-O as the key contributor of the aroma profile of several fruits such as pineapples [41], strawberries [42], cranberries [43] and melons [44]. The preliminary sensory tests (e.g., sniffing and vaping) on e-liquids 3-A, 4-A and 5-A, performed by both the panelists before GC-MS-O analytical sessions, allowed to appreciate a significant discrepancy between the flavor reported on the label and the overall flavor perceived. E-liquids 3-A and 4-A labels 'Davidoff' and 'Green USA mix' referred to tobacco brands

whilst e-liquid 5-A label reported 'cigar' flavor. In all three cases, the overall flavor coming from e-liquids vaporization should have simulated the characteristic notes of the tobacco leaves aroma (i.e., woody, leather). Instead, the qualitative description provided by both assessors highlighted that the overall e-liquids flavors were dominated by sweet and caramel-like notes with the only exception of e-liquid 5-A that in addition was characterized by distinct woody notes. GC/MS-O analysis of sample 3-A ('Davidoff' flavor) allowed to confirm the role of ethoxyethane in giving the formulation a characteristic sweet and pleasant flavor. Moreover, the odor-active compound found to be the key contributor to the caramel notes of the overall flavor was 2,3-butanedione (or diacetyl), whose relevance as a flavoring additive will be deeply discussed in Section 4. Similarly to samples 1-A and 2-A, other odorous stimuli perceived approximately at 17.6 and 20.4 min and resembling tobacco flavor were associated with low intensity chromatographic peaks and, as a result, the tentative attribution was not allowed. At this regard, it has been already highlighted in Tierney et al., 2015 that the majority of tobacco flavored liquids were found to contain confectionary flavor chemicals instead of tobacco extracts therefore it is likely that the flavor chemicals pattern (i.e., benzyl alcohol, vanillin, ethylacetate, maltol) included in the formulations for resembling tobacco flavor is not necessarily what is expected to be found in a tobacco extract [45]. Considerations made for sample 3-A are relevant also for sample 4-A ('Green USA mix' flavor). Ethoxyethane and diacetyl were also detected in sample 4-A and associated, similarly with sample 3-A, to sweet and caramel-like flavor notes respectively. In addition, ethoxybutane was identified and associated with sweet flavor notes. The attribution for other odorous stimuli perceived during the GC/MS run, approximately at 8.7, 11.6 and 17.6 min (the latter similarly with sample 3-A), was not successful due to low intensity chromatographic peaks. More specifically, in addition to tobacco-like flavor, hearbaceous and grass/mint notes were perceived by assessors and this perception was considered reliable taking into account that, at least in principle, the formulation 'Green USA mix' should have simulated menthol-tobacco cigarettes and its characteristic menthol and herbaceous flavor notes. A comprehensive list of flavoring additives was obtained for sample 5-A ('cigar' flavor). Ethoxyethane and 2-ethoxybutane (both perceived with intensity 2) were confirmed as key contributors for sweet flavor notes while diacetyl (perceived with intensity 3) responsible for the caramel-like flavor. An interesting GC-MS-O outcome, allowing us to characterize the odor profile of the sample 5-A in a more distinctive way, was the identification of three odor-active compounds, perceived with odor intensity ranging from 1 to 2: α-terpinene (woody, 16.3 min), α-phellandrene (woody, 18.3 min), and α-terpinolene (woody/pine, 19.4 min). They all are classified as flavoring agents by FEMA: the associated flavor profile varies from woody, fresh, citrus, and spice notes in the case of α-phellandrene to pine flavor notes in the case of α-terpinolene. Their inclusion in the liquid formulation is therefore related to the intention of enriching the overall flavor profile of the product with woody and pine flavor notes with the purpose to simulate as closely as possible the cigar flavor. Finally, the integration of sensory perception and GC-MS chromatographic data failed in the identification of the odor-active compound perceived by both evaluators as responsible for the tobacco-like and burnt flavor, similar to what was previously observed for the sample 3-A.


