2.3.2. GC-MS-O Analysis Conditions

The VOCs collected were analyzed using an air sampler-thermal desorber integrated system (UNITY 2™Markes International Ltd, Llantrisant, UK) connected to a gas chromatograph (7890 Agilent Technologies, Santa Clara CA, USA) equipped with an Olfactory Detection Port (ODP 3 Gerstel GmbH&Co, Mülheim an der Ruhr, Germany) and a single-quad mass spectrometer (5975 Agilent Technologies, Santa Clara CA, USA). The collection of VOCs onto the sorbent-pack focusing trap at −10◦C of the desorption system UNITY2™ was performed by connecting the Nalophan bag to the inlet port of the automated air sampling device. The cold trap was flash heated to 300 ◦C and the compounds were transferred via the heated transfer line (200 ◦C) to the GC column and to the ODP port. The chromatographic separation was performed on a HP5-MS capillary column (30m × 250μm × 0.25μm). Carrier gas (Helium) flow was controlled by constant pressure and equal to 1.7 ml/min. The GC oven temperature program was set as follows: from 37 ◦C up to 100 ◦C at 3.5 ◦C/min (ramp 1); and from 100 ◦C up to 250 ◦C at 15 ◦C/min (ramp 2). After the GC separation, the column flow was split into two parts (ratio 1:1), one part was connected to the MS detector and the other one to ODP. The transfer line connecting the GC column and MS detector was kept at 250 ◦C. The mass spectrometer was operated in electron impact (EI) ionization mode (70eV) in the mass range 20–250 m/z. The effluent from the capillary column was connected to the ODP port through an uncoated transfer line (deactivated silica capillaries), constantly heated to prevent compounds condensation. Two trained panelists, one male and one female (24 years old), were asked to sniff in the conical ODP simultaneously with the GC run, indicating exactly when they start and stop perceiving the odor and providing a qualitative description of the odor (using suitable descriptors) [36] and odor intensity based on an intensity scale from 0 (no odor perceived) to 4 (strong odor). Auxiliary air (make-up gas) was added to the GC effluent to prevent the assessors' nose mucous membranes drying, which may potentially cause discomfort, especially in extended analysis sessions. The panelists involved in the present study had previously been selected according to a standardized procedure used for the panel selection in Dynamic Olfactometry, the official methodology for odor emissions assessment standardized by a European technical law (EN 13725/2003) [39]. The standardized procedure provides for individuals with average olfactory perception sensitivity that constitute a representative sample of the human population. The screening was performed evaluating the response to the most used reference gas, 1-butanol. Only assessors who fulfilled predetermined repeatability and accuracy criteria were selected as panelists. The identification of flavoring additives and other VOCs in e-liquid formulation was performed by comparing the mass spectra obtained with those listed in the NIST library (Agilent Technologies). It was considered valid when the confidence rating of mass spectra comparison was superior or equal to 95%. The attribution was further confirmed using the retention times of authentic compounds. Before GC-MS-O sessions, panelists were asked to carry out preliminary sensory tests by sniffing and vaping the liquid formulations. This preliminary approach revealed to be useful in appreciating discrepancies between the flavors reported on e-liquid labels and the overall flavor perceived by panelists' noses and mouths (see Section 3.2 in results section).
