# Volatile Composition of Smoked and Non-Smoked Iranian Rice
## 2 Materials and Methods
### Chromatographic Analyses  
The rice volatile compounds were analyzed and identified using a Shimadzu GC-17A gas chromatograph connected with a Shimadzu QP-5050A mass spectrometer detector (Shimadzu Corporation, Kyoto, Japan). The GC-MS system consisted of a TRACSIL Meta.X5 (95% dimethylpolysiloxane and 5% diphenylpolysiloxane) column (60 m \(\times\) 0.25 mm, 0.25 \(\upmu\)m film thickness; Teknokroma S. Coop. C. Ltd, Barcelona, Spain). The carrier gas used to perform the analysis were helium, at a column flow rate of 0.6 mL\(\cdot\)min\({}^{-1}\), and a total flow of 6.0 mL\(\cdot\)min\({}^{-1}\) in a split ratio of 1:6. The oven program was as follows: (a) 80 \({}^{\circ}\)C for 0 min; (b) increase of 3 \({}^{\circ}\)C\(\cdot\)min\({}^{-1}\) from 80 \({}^{\circ}\)C to 210 \({}^{\circ}\)C, and hold for 1 min; (c) increase of 25 \({}^{\circ}\)C\(\cdot\)min\({}^{-1}\) from 210 \({}^{\circ}\)C to 300 \({}^{\circ}\)C, and hold for 3 min. The injector and detector temperatures were 230 \({}^{\circ}\)C and 300 \({}^{\circ}\)C, respectively.  
Three protocols were used to identify the rice volatile compounds: (1) retention indices and its correlation with those from the literature; (2) GC-MS retention times (authentic compounds); (3) mass spectra (authentic chemicals and NIST05 spectral library collection) [17]. Only fully identified compounds have been reported in this study.  
The relative abundance of the volatile compounds (%) was performed on a gas chromatograph, Shimadzu 2010, with a flame ionization detector (FID). The column and chromatographic conditions were those previously reported for the GC-MS analysis. The injector temperature was 200 \({}^{\circ}\)C and nitrogen was used as carrier gas (1 mL\(\cdot\)min\({}^{-1}\)). The relative abundance was obtained from electronic integration measurements using flame ionization detection (FID). Anethole (1000 mg\(\cdot\)L\({}^{-1}\)) was added as internal standard at the beginning of the distillation procedure to simulate the behavior of volatile  
\begin{table}
\begin{tabular}{c c c c c c} \hline
**Sample Code** & **Cultivar** & **Comments** & **Province** & **City** & **Location** \\ \hline R12 & Tarem & Smoked & Mazandaran & Fereydun kenar & North \\ R13 & Lenjun & - & Isfahan & Lenjan & Center \\ R14 & Tarem & - & Lorestan & Borujerd & West \\ R15 & Shamshiri & - & Ilam & Chardaval & West \\ \hline \end{tabular}
\end{table}
Table 1: _Cont._compounds; this chemical was used as internal standard after checking that it was absent in herbs, it separates well from other volatiles, it possesses similar FID and MS response factors to most of the volatiles in the aromatic herb essential oil, it is stable at high temperatures and does not react with water. This internal standard, anethole, was used to normalize the area of all the rice volatiles.  
The volatile composition analysis was run in triplicate and results were expressed as a percentage of the total area represented by each one of the volatile compounds [16; 18]. Thus, GC-MS was used for identification of the volatile compounds while GC-FID was used for establishing the relative abundance of the volatiles. This combination of GC-MS and GC-FID has been successfully used for the identification and relative abundance (%) or quantification (ug\(\cdot\)L\({}^{-1}\) or ug\(\cdot\)kg\({}^{-1}\)) of volatile compounds in several matrices, such as aromatic herbs [18; 19], wild bitter almonds (_Amygdala scoparia_) [20], and _Origanum majorana_[21], among others.