## 2 Results and Discussion
### Effect of Germination on the Volatile Profile of IWG
#### 2.2.3 MHE Optimization  
The first step for optimization of the MHE was evaluating the amount of sample required. The area of volatile compounds should decrease by at least 5% between each extraction to achieve \(\beta\) values between 0.4 and 0.95 [38]. Values below 0.4 indicate that the analyte was fully extracted in the first extraction, making MHE unnecessary. Values above 0.95 suggest saturation of the HS due to high analyte concentrations, in which case the sample amount should be reduced to use MHE effectively. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Trial** & **Temp (\({}^{\circ}\)C)** & **Time (min)** & **Total Area** \\ \hline
1 & \(-\)1 (39.40) & \(-\)1 (34.36) & 669,149,921 \\
2 & 1 (60.60) & \(-\)1 (34.36) & 733,292,935 \\
3 & \(-\)1 (39.40) & 1 (55.64) & 750,239,779 \\
4 & 1 (60.60) & 1 (55.64) & 845,258,191 \\
5 & \(-\)1.41 (35.00) & 0 (45.00) & 656,168,788 \\
6 & 1.41 (65.00) & 0 (45.00) & 846,707,555 \\
7 & 0 (50.00) & \(-\)1.41 (30.00) & 639,981,632 \\
8 & 0 (50.00) & 1.41 (60.00) & 808,548,691 \\
9 & 0 (50.00) & 0 (45.00) & 711,222,561 \\
10 & 0 (50.00) & 0 (45.00) & 734,532,059 \\
11 & 0 (50.00) & 0 (45.00) & 672,019,319 \\
12 & 0 (50.00) & 0 (45.00) & 669,149,921 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Experimental conditions (coded/real values) and response for the optimization of the extraction conditions of volatile compounds from IWG flour by HS-SPME. Figure 3: Response surface (**A**) and contour plots (**B**) of the impact of time and temperature on the total area of volatiles from IWG. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Source of Variation** & **Sum of Squares** & **Degrees of Freedom** & **Mean of Squares (MS)** & **F Cal** & **F Tab** \\ \hline Regression (R) & 5.03 \(\times\) 10\({}^{16}\) & 5 & 1.01 \(\times\) 10\({}^{16}\) & 12.39 & 5.05 \\ Residue (r) & 4.06 \(\times\) 10\({}^{15}\) & 5 & 8.12 \(\times\) 10\({}^{14}\) & & \\ Lack of fit & 2.06 \(\times\) 10\({}^{15}\) & 3 & 6.88 \(\times\) 10\({}^{14}\) & 0.69 & 19.6 \\ Pure error & 1.97 \(\times\) 10\({}^{15}\) & 2 & 9.99 \(\times\) 10\({}^{14}\) & & \\ Total & 5.44 \(\times\) 10\({}^{16}\) & 10 & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Analysis of variance (ANOVA) including models and R\({}^{2}\) for the extraction of volatile-form IWG by HS-SPME. Determining the amount of sample required to promote the decay for a multianalyte quantitation over a wide range of concentrations is a challenging task. For this evaluation, 600, 800, and 1000 mg masses were used and six extractions were performed. The sample size under study should be sufficient to release the minimum amount of analyte needed to match the method's sensitivity and precision without saturating the HS [39]. The sample size of 600 and 800 mg were not sufficient to provide the exponential decay of the analytes (Figure 4). For some important compounds widely reported in cereal flour, such as hexanal, the area increased during the extraction using 600 and 800 mg (Figure 4A,B). However, the decay was achieved when 1000 mg of the sample was used (Figure 4C). Comparing the signals obtained for the three sample amounts evaluated (Figure 4D), it was noticed that the signal for hexanal using 600 and 800 mg was significantly lower in comparison with the signal using 1000 mg. This reduced signal could result in the absence of decay in the area during multiple extractions. For this reason, 1000 mg was selected to optimize the MHE protocol. A total of fifty-eight compounds were identified and their \(\beta\) values are shown in Table 4. \(\beta\) values ranging from 0.51 to 0.90 are within the limit recommended. \(\beta\) values below 0.4 indicate depletion of the analytes with successive extractions. On the other hand,  
Figure 4: Decay after 6 extractions (**A–C**) and overlaid signals of the first extraction (**D**) for hexanal using 600, 800, and 1000 mg of sample. values higher than 0.95 imply that the amount in the vial remains constant, and the area of the analyte after successive extractions is the same [38].