2.4. Determination of Volatile Compounds by GC-MS

The extraction of volatile compounds was carried out according to previous research [23]. Briefly, 100 randomly selected grapes were deseeded and ground under liquid nitrogen. Fifty grams of the obtained powder was mixed with 1 g polyvinylpolypyrrolidone (PVPP), and the mixture was macerated at 4 °C for 3 h under a nitrogen atmosphere. Then, the mixture was centrifuged at 8000 rpm (4 °C) for 20 min. The clear juice or wine was filtered twice through 0.45 μm nylon 66 organic membranes. For volatile compound determination, 10 mL of wine with 40 μL of 2-octanol as an internal standard (0.32 g/L dissolved in ethanol, Sigma-Aldrich (Shanghai, China) was blended with 2 g NaCl in a 20 mL sample vial containing a magnetic stirrer. The vial was tightly capped with a PTFE-silicon septum for further analysis.

GC-MS analyses were performed using a Thermo-Finnigan Trace 2000 gas chromatograph (Thermo Finnigan, Waltham, MA, USA) with an HP-INNWAX column (60 m, 0.25 mm I.D., 0.25 μm; Agilent, Shanghai, China). The solid-phase micro-extraction fiber (SPME, DVB/CAR/PDMS 2CM, 50/30-μm) was heated at 250 °C for 2 h. The vial containing the sample (wine) was extracted in a 40 °C water bath for 30 min while being stirred and then desorbed at 230 °C for 3 min into the splitless injection port of the GC-MS instrument. Helium was used as the carrier gas (1 mL/min). The chromatographic conditions consisted of an initial oven temperature of 40 °C for 2.5 min, increasing the temperature to 150 °C at a rate of 5 °C/min, increasing to 220 °C at a rate of 3 °C/min and holding for 30 min. The temperatures of the ion source and MS transfer line were 250 °C and 280 °C, respectively. The scan range was 33–450 m/z with electron impact (EI) mode at 70 eV.

The NIST 2002 mass spectroscopy library (National Institute of Standards and Technology, Gaithersburg, MD, USA) and the retention times of the authentic standards were used to identify the volatile compounds. Quantification was performed using the peak areas on the total ion chromatogram. The concentrations of the volatile compounds were calculated based on their calibration curves (the relative response rate was greater than 98%), which were built by plotting the area ratio of the target compounds to the internal standard against the concentration ratio [24]. All determinations were performed in triplicate.