# SPME Method Optimized by Box-Behnken Design for Impact Odorants in Reduced Alcohol Wines
## 2 Materials and Methods  
### Chemicals  
Ethyl butyrate, ethyl-2-methyl butyrate, ethyl-3-methyl butyrate, isoamyl acetate, 3-methyl-1-butanol, ethyl hexanoate, ethyl-s-lactate, (z)-3-hexenol, methyl octanoate, ethyl octanoate, propanoic acid, linalool, methyl decanoate, ethyl decanoate, isoamyl octanoate, 3-(methylthio)-1-propanol, \(\beta\)-phenyl ethyl acetate, ethyl dodecanoate, geraniol, \(\beta\)-phenyl ethanol, octanoic acid, decanoic acid, and vanillin were purchased from Fulka (Buchs, Switzerland). The analytes' identification numbers for the Box-Behnken design, odors and olfactory thresholds, and boiling points of each compound are presented in Table 1. Ethanol (VWR Prolabo, Fontenay Sous Bois, France), L-(+) tartaric acid (Sigma, Steinheim, Germany), and potassium hydrogen tartrate (BHD Chemicals Ltd., Poole, UK) were used for the preparation of the model wine solutions. The deionized water was obtained from a Milli-Q mixed bed resin system (18 M\(\Omega\)/cm, 25 \({}^{\circ}\)C).  
The internal standards comprising of 2-octanol, 4-methyl-2-pentanone, and deuterated isotopes of ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, and phenol were used for the preparation of the calibration curves for the target compounds (Tables S3-S5). The deuterated ethyl esters were prepared in accordance with the published methods [19] and the deuterated phenol was obtained from the Polymer Source (Dorval, QC, Canada). An internal standard mix containing 2-octanol (1.94 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), 4-methyl-2-pentanone (99 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), d5-ethyl butyrate (8.08 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), d5-ethyl hexanoate (9.12 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), d5-ethyl octanoate (3.78 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), d5-ethyl decanoate (3.74 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), and d6-phenol (20.26 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) was prepared in ethanol and stored at \(-80\)\({}^{\circ}\)C prior to use.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Code** & **Compounds** & **Odors 1** & **OT 1 (\(\mathrm{\SIUnitSymbolMicro g}\)/L)** & **BP (\({}^{\circ}\)C)** \\ \hline
1 & ethyl butyrate & apple & 20 & 121 \\
2 & ethyl-2-methyl butyrate & apple & 1–18 & 138 \\
3 & ethyl-3-methyl butyrate & sweat, acid, rancid & 33.4 & 134 \\
4 & isoamyl acetate & banana & 30 & 130 \\
5 & 3-methyl-1-butanol & whiskey, malt, burnt & 30,000 & 132 \\
6 & ethyl hexanoate & apple peel, fruit & 2–14 & 167 \\
7 & ethyl-s-lactate & fruit, milk 2 & 154,000 & 154 \\
8 & (z)-3-hexenol & green (cut grass) & 400 & 156 \\
9 & methyl octanoate & waxy, apple peel 2 & - & 192 \\
10 & ethyl octanoate & fruit, fat & 2–5 & 207 \\
11 & propanoic acid & pungent, rancid, soy & 8100 & 141 \\
12 & linalool & flower, lavender & 25.2 & 198 \\
13 & methyl decanoate & wax, soap, fruit 2 & - & 108 \\
14 & ethyl decanoate & grape & 200 & 245 \\
15 & isoamyl octanoate & wax, soap, pear 2 & - & 267 \\
16 & 3-(methylthio)-1-propanol & sweet, potato & 1000 & 90 \\
17 & \(\beta\)-phenyl ethyl acetate & rose, honey & 250 & 229 \\
18 & ethyl dodecanoate & wax, soap 2 & - & 269 \\
19 & geraniol & rose, geraniom & 30 & 230 \\
20 & \(\beta\)-phenyl ethanol & honey, rose & 10,000–14,000 & 219 \\
21 & octanoic acid & sweat, cheese & 500 & 240 \\
22 & decanoic acid & rancid, fat & 1000 & 268 \\
23 & vanillin & vanilla & 200 & 285 \\ \hline \hline \end{tabular}  
* OT: odor threshold; BP: boiling point. 1 From [PERSON] and [PERSON] [20] except where specified. 2 From [PERSON], et al. [21].  
\end{table}
Table 1: The compound identification for Box-Behnken design, odors and olfactory thresholds, and boiling point.