# To What Extent Are the Effects of UV Radiation on Grapes Conserved in the Resulting Wines?
## 4 Materials and Methods
### Grape Sampling, Winemaking, and Wine Analysis  
For each radiation regime and replicate, grapes were collected at harvest (5 September) and analyzed for phenolic compounds and volatile organic compounds (VOCs), as described in a previous study [9]. Additional grapes were separately collected for winemaking. Grapes were destemmed and crushed, and alcoholic fermentation was performed [23]. Around 3 kg of crushed grapes were introduced into 2.5 L glass bottles. Potassium metabolsulfite (0.09 g kg\({}^{-1}\)) was added to the samples to give a final total SO\({}_{2}\) concentration of 50 mg L\({}^{-1}\), and then mucus were inoculated with 0.2 g kg\({}^{-1}\) of commercial _Saccharomyces cerevisiae_ r.f. _hyams_ (Enartis, Trecate, Italy). The must was fermented at a controlled temperature of 25 \({}^{\circ}\)C. The alcoholic fermentation finished when reducing sugars were below 2.5 g L\({}^{-1}\) (two weeks after yeast inoculation). Then, the wine was separated from the seeds and skins by pressing, and the wine analysis was performed. Color intensity (CI), hue and total polyphenol index (TPI) were analyzed according to official methods [41]. Total phenols, antioxidant capacity, the bulk levels of UV-absorbing compounds (UVAC), individual phenolic compounds, and VOCs, were analyzed as in berry skins in previous studies [9; 14]. Total phenols were determined using the Folin-Ciocalteu reagent and expressed as the gallic acid equivalents (GAE). The antioxidant capacity was measured by generating the radical cation 2,2\({}^{\prime}\)-azinobis(3-ethylbenzothazoline-6-sulfonic acid) (ABTS\(\bullet\)+), and expressed in terms of Trolox equivalents (TE). The bulk level of UV-absorbing compounds (UVAC) was measured as the area under the absorbance curve in two wavelength intervals, 280-315 and 280-400 nm, corresponding to UV-B and the sum of UV-B and UV-A (AUC\({}_{\rm 280-315}\) and AUC\({}_{\rm 280-400}\), respectively), using a Perkin Elmer \(\lambda\)35 spectrophotometer (Perkin Elmer, Wilton, CT, USA). Individual phenolic compounds were analyzed by UPLC/LC-MS (Waters Acquity UPLC system, Waters Corporation, Milford, MA, USA). Solvents were: A, water/formic acid (0.1%), and B, acetonitrile with 0.1% formic acid. The gradient program employed was: 0-7 min, 99.5--80% A; 7-9 min, 80--50% A; 9-11.7 min, 50--0% A; 11.7-15 min, 0--99.5% A. The UPLC system was coupled to a micrOTOF-QII-ESI-MS/MS high-resolution mass spectrometer (Bruker Daltonics, Bremen, Germany) controlled by the Bruker Daltonics Data Analysis software. The electrospray (ESI) source was operated in positive or negative mode, in the range of \(m/z\) 120 and 1505. The optimized conditions of the ESI source were as follows: capillary potential 4 kV, ESI source temperature 180 \({}^{\circ}\)C, desolvation temperature 200 \({}^{\circ}\)C, gas flow 9 L min\({}^{-1}\); nebulizer gas 3.5 bar and 25 \({}^{\circ}\)C. LC-MS and MS/MS were performed operating in continuum mode. The spectra were acquired at two scans per second. The fragmentor voltage for MS/MS acquisition mode was 35 eV. The identity assignation of compounds was carried out by combining different information: retention time, UV-Vis data, MS spectra, MS/MS fragmentation patterns of peaks of available pure compounds, and literature data [42]. For quantification, DAD chromatograms were extracted at 520 nm for anthocyanins and 324 nm for the other compounds; the calibration curves of the respective standards were used.