# Chemical and Sensory Characterization of Vidal Icewines Fermented with Different Yeast Strains
## 2 Materials and Methods
### Analysis of Volatile Aroma Compounds  
Headspace solid-phase microextraction coupled with comprehensive 2-dimensional gas chromatography and time-of-flight mass spectrometry (HS-SPME-GC\(\times\)GC-TOFMS) was employed to determine the volatile profile of the icewine samples, and each sample was analyzed in 3 replicates. Based on previously described methods with slight modifications [23], 5 mL of icewine were placed into 20 mL glass with a silicon septum and saturated with 1.5 g of NaCl. Internal standard mixture (10 uL; IS1: n-hexyl- d13-alcohol, 403.76 uB/L; IS2: 2-methoxy-d3-phenol, 197.6 uB/L; IS3: menthyl acetate, 20.08 uB/L) was added as an internal standard used for the semi-quantification of aroma compounds. A MultiPurpose autosampler (Gerstel GmbH and Co. KG, Mulheim an der Ruhr, Germany) with a 50/30 uMivilbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber (2 cm; Supelco Inc., Bellefonte, PA, USA) was used to extract the volatile compounds from the headspace of the sample vial based on the previous work with slight modifications. The sample was equilibrated at 50 \({}^{\circ}\)C for 5 min and then extracted for 45 min under stirring (250 rpm). After extraction, the fiber was inserted into the gas chromatograph injection port (250 \({}^{\circ}\)C) and desorbed for 5 min (splitless) to GC \(\times\) GC-TOFMS analysis.  
A LECO Pegasus 4D GC \(\times\) GC-TOFMS instrument (LECO Corporation, St. Joseph, MI, USA) equipped with an Agilent 7890B gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) was used in GC \(\times\) GC-TOFMS analysis. A polar/moderately polar column set was optimized for the GC \(\times\) GC separation. The first dimension (1D) column was DB-FFAP column (60 m \(\times\) 0.25 mm i.d. and 0.25 um film thickness, Agilent Technologies Inc., Santa Clara, CA, USA), and the second dimension (2D) column was Rxi-17 Sil MS Cap. column (1.5 m \(\times\) 0.25 mm i.d.and 0.25 um film thickness, Restek Technologies Inc, Bellefonte, PA, USA). The oven temperature of the first column was held at 45 \({}^{\circ}\)C for 2 min, ramped at 4 \({}^{\circ}\)C/min to 230 \({}^{\circ}\)C, held for 15 min. The temperature of the second column was held at 40 \({}^{\circ}\)C for 2 min, ramped at 5 \({}^{\circ}\)C/min to 250 \({}^{\circ}\)C, held for 5 min. A quad-jet, dual-stage thermal modulator was equipped between the 1D and 2D columns. The modulation period as set for 4 s with a 1 s hot pulse time. Helium (99.9995% purity) was used as the carrier gas at a constant flow of 1.0 mL/min. For the TOFMS system, the temperatures of the transfer line and ionization sources were 280 and 230 \({}^{\circ}\)C, respectively. Electron impact mass spectra were recorded at 70 eV, and the acquisitions were performed over an m/z scan range of 35-400 amu.  
LECO ChromaTOFTM Workstation (version 4.44) was used for all acquisition control and data processing. Automated peak detection and spectral deconvolution were employed. The minimum value for the signal to noise (S/N) ratio necessary to record a chromatographic peak was set as 50 in GC \(\times\) GC. The positive identification of the compounds was achieved by comparing the retention data and mass spectra of the standard compounds. For unavailable standards, tentative identification of the compounds was achieved by comparing their experimental retention indices (I) with the retention indices reported in the scientific literature (IT). A compound was considered to be tentatively identified if the similarity between mass spectrometric information of each chromatographic peak and the National Institute of Standards and Technology (NIST) mass spectra library was at least 75%, and the difference between I and IT did not exceed 30 units. Semi-quantitativeanalysis was performed in triplicate, and average values of concentration were used in further data elaboration.