# Identification of Rotundone as an Important Contributor to the Flavor of Oak-Aged Spirits
## Materials and Methods
### Materials  
Toasted oak was obtained from Oak Chips Inc. (Waverly, OH, USA). _Cyrpus rotundus_ \"whole herb\" (dried root) was from Chinese Herbs Direct (Torrance, CA, USA). Ground white peppercorn, _Piper nigrum_ (Spice Islands Trading Co., San Francisco, CA, USA), was purchased locally (Champaign, IL, USA). All spirits were purchased from a local liquor store (Binny's Beverage Depot, Champion, IL, USA). These included the following bourbons: Jim Beam Bourbon (4 year), Jim Beam Black Bourbon (8 year), Jim Beam Signature Craft Bourbon (12 year), Bulleti Bourbon (at least 6 year), Bulleti Bourbon 10 year (10 year), Elizabeth Craig Bourbon (12 year), W.L Weller Bourbon (12 year). Other aged spirits were Jack Daniels Tennessee Whiskey (at least 4 year), Johnnie Walker Black Label Scotch Whiskey (at least 12 year), Bulleti Rye Whiskey (at least 4 year), Appleton Estates Extra Rum (12 year), Don Julio Arejo Tequila (18 month), \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Method** & **Target** & **Labeled IS** & **Mass IS (\(\mu\)g)** & **Sample (mL)** &
\begin{tabular}{c} **Response Factor, R\({}_{f}\)** \\ _(intarget vs. ion\({}_{IS}\))_ \\ \end{tabular} \\ \hline \multirow{8}{*}{SPME (3.5.1)} & acetal & [\({}^{2}\)H\({}_{8}\)]-ethyl acetate & 243 & 1.0 & 0.521 (_103 vs. 96_) \\  & ethyl butanoate & [\({}^{2}\)H\({}_{5}\)]-ethyl butanoate & 1.51 & 1.0 & 0.771 (_88 vs. 93_) \\  & ethyl hexanoate & [\({}^{2}\)H\({}_{5}\)]-ethyl hexanoate & 2.50 & 1.0 & 0.831 (_88 vs. 93_) \\  & ethyl octanoate & [\({}^{2}\)H\({}_{5}\)]-ethyl octanoate & 2.10 & 1.0 & 0.797 (_88 vs. 93_) \\  & ethyl & [\({}^{2}\)H\({}_{5}\)]-ethyl 3-methyl & 0.797 & 1.0 & 0.834 (_88 vs. 93_) \\  & 3-methylbutanoate & [\({}^{2}\)H\({}_{5}\)]-ethyl vanillate & 0.168 & 1.0 & 0.954 (_196 vs. 201_) \\  & 3-methylbutyl acetate & 3-methylbutyl & 3.89 & 1.0 & 0.834 (_87 vs. 90_) \\  & & [\({}^{2}\)H\({}_{3}\)]-acetate & & & \\  & 2-phenylethyl acetate & [1,2-\({}^{13}\)C\({}_{2}\)]-2-phenylethyllyl & 0.111 & 1.0 & 0.932 (_104 vs. 106_) \\  & 2-phenylethanol & [\({}^{13}\)C\({}_{2}\)]-2-phenylethylhanol & 41.4 & 1.0 & 1.00 (_122 vs. 124_) \\  & \(\beta\)-damascenone & [\({}^{2}\)H\({}_{4}\)]-\(\beta\)-damascenone & 0.0352 & 1.0 & 0.468 (_190 vs. 194_) \\ \hline \multirow{8}{*}{DSE (3.5.2)} & (\(E\))-whiskey lactone & [\({}^{2}\)H\({}_{2}\)]-(\(E\))-whiskey lactone & 3.37 & 1.0 & 0.827 (99 vs. 101) \\  & (\(Z\))-whiskey lactone & [\({}^{2}\)H\({}_{2}\)]-(\(Z\))-whiskey lactone & 3.57 & 1.0 & 0.546 (99 vs. 101) \\  & \(\gamma\)-nonalactone & [\({}^{2}\)H\({}_{2}\)]-\(Y\)-nonalactone & 0.204 & 1.0 & 1.11 (85 vs. 87) \\  & guaiacol & [\({}^{2}\)H\({}_{3}\)]-gauaicol & 5.35 & 10.0 & 1.02 (124 vs. 127) \\  & \(p\)-cresol & [\({}^{2}\)H\({}_{8}\)]-p-cresol & 0.306 & 10.0 & 0.552 (107 vs. 113) \\  & 4-ethylphenol & [\({}^{2}\)H\({}_{5}\)]-4-ethylphenol & 2.30 & 10.0 & 0.845 (122 vs. 127) \\  & syringol & [\({}^{2}\)H\({}_{3}\)]-syringol & 13.3 & 10.0 & 0.933 (154 vs. 157) \\  & eugenol & [\({}^{2}\)H\({}_{3}\)]-eugenol & 2.40 & 10.0 & 0.611 (164 vs. 167) \\  & (\(E\))-isoeugenol & [\({}^{2}\)H\({}_{3}\)]-(\(E\))-isoeugenol & 2.44 & 10.0 & 0.932 (164 vs. 167) \\  & vanillin & [\({}^{2}\)H\({}_{3}\)]-vanillin & 24.6 & 10.0 & 0.974 (152 vs. 155) \\  & syringaldehyde & [\({}^{2}\)H\({}_{3}\)]-syringaldehyde & 175 & 10.0 & 0.611 (182 vs. 185) \\  & rotundone & [\({}^{2}\)H\({}_{4}\)]-rotundone & 0.0104 & 10.0 & 0.998 (218 vs. 206) \\ \hline \multirow{2}{*}{Direct injection (3.5.3)} & 2-methyl-1-propanol & [\({}^{2}\)H\({}_{7}\)]-2-methyl-1-propanol & 15.0 & 0.10 & 0.956 (_74 vs. 81_) \\  & 2-methyl-1-butanol & [\({}^{2}\)H\({}_{11}\)]-3-methyl-1-butanol & [\({}^{2}\)H\({}_{11}\)]-3-methyl-1-butanol & 15.8 & 0.10 & 0.963 (57 vs. 62) \\  & 3-methyl-1-butanol & & butanol & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Calibration table: target analytes, labeled internal standards, IS spike masses, sample volumes, MS ions, and response factors used for SIDA of whiskey. and Milagro Tequila (unaged).