# Bottle Aging Affected Aromatic and Phenolic Wine Composition More than Yeast Starter Strains
## 2 Materials and Methods
### Determination of Wine Phenolic Compounds by HPLC-DAD
#### 2.4.2 Analysis of Phenolic Compounds by HPLC-DAD  
Phenolic compounds were analyzed according to [PERSON] et al. [24] in an Agilent 1260 Infinity II chromatograph, equipped with a diode array detector (DAD). Samples were filtered and injected on a Licrospher(r) 100 RP-18 reversed-phase column (250 \(\times\) 4.0 mm; 5 \(\upmu\)m packing; Agilent) with pre-column Licrospher(r) 100 RP-18 (4 \(\times\) 4 mm; 5 \(\upmu\)m packing; Agilent), both at 40 \({}^{\circ}\)C. A flow rate of 0.630 mL/min was used. For the analysis of anthocyanins, 10 \(\upmu\)L of each wine sample was injected. Elements used were: (A) acetonitrile/water/formic acid (3:88.5:8.5, \(v/v/v\)), and (B) acetonitrile/water/formic acid (50:41.5:8.5, \(v/v/v\)). For the analysis of non-anthocyanin phenolic compounds, the injection volume was 20 \(\upmu\)L. Elements were: (A) acetonitrile/water/formic acid (3:88.5:8.5, \(v/v/v\)); (B) acetonitrile/water/formic acid (50:41.5:8.5, \(v/v/v\)), and (C) methanol/water/formic acid (90:1.5:8.5, \(v/v/v\)).  
The identification of phenolic compounds was carried out according to the retention times of available pure compounds and the UV-Vis data obtained from authentic standards and/or published in previous studies [25]. For the quantification, DAD chromatograms were extracted at 520 nm (anthocyanins), 360 nm (flavonols), 320 nm (hydroxycinnamic acids and stilbenes), and 280 nm (gallic acid and flavanols) and the calibration graphs of the respective standards (R\({}^{2}\) > 0.99) were used. When a standard was not available, the quantification was made with the calibration graph of the most similar compound. Hence, malvidin-3-O-glucoside was used for anthocyanins, quercetin-3-O-glucoside for flavonoids, _trans_-caftaric acid for free hydroxycinnamic acids and the corresponding tartaric esters, catechin for procyanidins B1 and B2, epicatechin for epigallocatechin, and _trans_-piceid and _trans_-resveratrol for their respective _cis_ isomers. The results of the phenolic compounds found in the wines at the end of MLF, and after 6 and 9 months are shown by chemical families, with the exception of gallic acid, which was the only hydroxybenzoic acid found in the wines, as the sum total of the individual compounds: total anthocyanins (sum of delphinidin-3-glc, cyanidin-3-glc, petunidin-3-glc, penoidin-3-glc, malvidin-3-glc, delphinidin-3-acglc, cyanidin-3-acglc, petunidin-3-acglc,peonidin-3-acglc, malvidin-3-acglc, delphinidin-3-cmglc, cyanidin-3-cmglc, petunidin-3-cmglc, penoidin-3-cmglc, malvidin-3-cis-cmglc, malvidin-3-trans-cmglc, malvidin-3-cfglc, vitisin A, and vitisin B; abbreviations: glc, glucoside; acglc, acetylglucoside; cmglc, _trans_-_p_-coumaroylglucoside; and cfglc, caffeoylglucoside); total flavonols (sum of myriceint-3-gal, myriceint-3-glc, myriceint-3-glc, quercetin-3-glc, laricitrin-3-glc, kaempfer-3-gal, kaempfer-3-glc, kaempfer-3-glc, kaempfer-3-glcu, isorhamnetin-3-glc, syringeint-3-glc, free-myricetin, free-quercetin, free-laricitrin, free-kaempfer, free-isorhamnetin, and free-syringetin; abbreviations: gal, galactoside; glcU, glucuronide; and glc, glucoside); total flavonols (sum of epigallocatechin, catechchin, epicatechin, procyanidin B1, and procyanidin B2); total hydroxycinnamic acids (sum of _trans_-caftaric acid, _trans_+_cis_-coutaric acids, caffeic acid, _trans_-fertaric acid, p-coumaric acid, and ferulic acid), and total stitbenes (sum of _trans_-piceid, _cis_-piceid, _trans_-resveratrol, and _cis_-resveratrol).  
Since viinifications for each of the Sc strain tested were performed in duplicate, the results for phenolic compounds are the average of the analyses of two samples (n = 2).