# The Influence of UV on the Production of Free Trepenes in _Vitis vinifera_ cv. [PERSON]
## 2 Materials and Methods
### Headspace Solid Phase Microextraction Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) Analysis  
First, 50 g of grape sample were ground into powder in liquid nitrogen. Then, 5 g of the sample powder were mixed with 30 mL of extraction solution (5 g/L of TA, 5 g/L of PVPP, 0.5 g/L of sodium sulphite, pH 3.2) and shaken for 24 h in a temperature-controlled incubator (ZWYR-240, Labwit Scientific, Ashwood, VIC, Australia) at 100 rpm and 22 \({}^{\circ}\)C. After centrifugation at 9000 rpm for 15 min at 20 \({}^{\circ}\)C, the supernatant was filtered through a 0.45 \(\upmu\)m nylon syringe filter (Thermo Scientific, Waltham, MA, USA). Then, 5 mL of the filtrate were transferred to a 20 mL gas chromatography (GC) sampling vial and mixed with 1 g of NaCl and 20 \(\upmu\)L of internal standard \(\beta\)-cedrene (2 mg/L). A polydimethylsiloxane/divinylbenzene (PDMS/DVB) 65 \(\upmu\)m solid-phase microextraction (SPME) fibre (Supelco, Bellefonte, PA, USA) was exposed to the headspace of the GC vial for 1 h at 45 \({}^{\circ}\)C with agitation before desorbing in a GC inlet.  
Figure 1: Experimental design for the ultraviolet (UV) attenuation trial at different phenological stages of grapevine development. The treatments were as follows: (i) control: grape bunches were naturally exposed to sunlight; (ii) grape bunches were enclosed by polyethylene terephthalhalate glycol (PETG) or polycarbonate (PC) film from pre-veraison to harvest (TW PETG/TW PC); (iii) grape bunches were enclosed by PETG or PC from pre-veraison to full veraison (TV PETG/TV PC); (iv) grape bunches were enclosed by PETG or PC from full veraison to intermediate ripe (TI PETG/TI PC); (v) grape bunches were enclosed by PETG or PC from intermediate ripe to harvest period (TH PETG/TH PC).  
An Agilent PAL multipurpose sampler connected to an Agilent 6890-5973 gas chromatography-mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) was used to carry out the analysis. The GC was equipped with a J&W DB-5 ms capillary column (Agilent Technologies 30 m \(\times\) 0.25 mm, 0.25 mm film). The carrier gas was helium (99.999% purity, BOC, Adelaide, Australia) at a constant flow rate at 1.0 mL/min. A resilanised borosilicate glass SPME inlet liner (Supelco, 6.5 mm o.d., 0.75 mm i.d., 78.5 mm long) was installed in the GC inlet and held at 220 \({}^{\circ}\)C [25]. The SPME fibre was desorbed for 10 min in the pulsed splitless mode at 25 psi for 30 s. The mass spectrometry was operating at scan/selected ion monitoring (SIM) mode at gain factor 15 to maximise the signal. Scan mode ranged from \(m\)/\(z\) 35 to 280. SIM mode recorded common sesquiterpene ions, namely \(m\)/\(z\) 105, 133, 147, 161 and 204, to facilitate the identification of sesquiterpenes at low concentrations. Quantification of all terpen delivered to the GC via SPME sampling was based on the target ion response areas [26]. A mixed alkane standard with a range of C7-C30 was analysed to calculate the retention index (RI). Sesquiterpenes were tentatively identified using the MassFinder 4 software with its built-in terpenes library (Hochmuth Scientific Consulting, Hamburg, Germany) by comparing the RI and mass spectra. Quantification was conducted using standard calibration curves of terpene standards (\(\alpha\)-terpineol, linalool, geraniol and geranylacetone). Identified terpenes without authentic chemical standard were semi-quantified by a known terpene standard. Sesquiterpenes and norisoprenoids were semi-quantified with the internal standard as described previously [27].