# HS-SPME Gas Chromatography Approach for Underivatized Acrylamide Determination in Biscuits
## 3 Results and Discussion
### Optimization of SPME Extraction Conditions  
HS-SPME conditions using CAR/PDMS fiber were optimized by evaluating the effect of three factors on AA extraction: temperature (T), propanol volume (V), and fiber exposure time (t). The experimental plan with 13 experimental runs generated using a Box-Behnken design is shown in the Table 2. The results of analysis of variance of the designed experiments were obtained using ANOVA model comprising all main effects and their interactions (Table 3). According to ANOVA, only the main effects from the propanol volume (X\({}_{\text{V}}\)) and the fiber exposure time (Xt) were statistically significant (\(p<0.05\)), while no interaction between factors (X\({}_{\text{T}}\)X\({}_{\text{V}}\), X\({}_{\text{T}}\)X\({}_{\text{t}}\), and X\({}_{\text{V}}\)X\({}_{\text{t}}\)) was found to be significant. Temperature (X\({}_{\text{T}}\)) did not present a significant impact on the extraction conditions (\(p>0.05\), Table 3). Under the tested conditions, the propanol volume (V) is the most significant variable. When using only water, the chromatographic areas were low (\(<\)1.5 \(\times\) 10\({}^{4}\), Figure 3). However, higher AA extractability was observed when higher proportions of propanol were used (lowest \(p\)-value, negative \(b\)-coefficient). Nevertheless, propanol alone resulted in low GC chromatographic areas. Due to AA high solubility in water (390 g/L), its extractability to the headspace is low, explaining the low AA peak areas determined when only water is \begin{table}
\begin{tabular}{c c c} \hline \hline
**Variable** & \(b\)**-Coefficient** & \(p\)**-Value** \\ \hline Model & - & **0.016** \\ Intercept & 516,900 & \\ X\({}_{\text{T}}\) & 6299 & 0.442 \\ X\({}_{\text{V}}\) & \(-\)467,400 & **0.007** \\ X\({}_{\text{t}}\) & \(-\)411,900 & **0.016** \\ X\({}_{\text{T}}\)X\({}_{\text{V}}\) & \(-\)83,380 & 0.257 \\ X\({}_{\text{T}}\)X\({}_{\text{t}}\) & 42,320 & 0.562 \\ X\({}_{\text{R}}\)X\({}_{\text{t}}\) & 88,900 & 0.227 \\ \hline \hline \end{tabular} The notation “-” represents no \(b\)-coefficient. Bold numbers refer to significant variation sources (\(p<0.05\)) for both model and parameters. The operating conditions are temperature (T, \({}^{\text{C}}\)), propanol volume (V, mL), and the fiber exposure time (t, min). X\({}_{\text{T}}\)—effect of temperature, X\({}_{\text{V}}\)—effect of propanol volume, X\({}_{\text{t}}\)—effect of the interaction time, X\({}_{\text{T}}\)X\({}_{\text{V}}\)—interaction of the effects of temperature and propanol volume, X\({}_{\text{T}}\)X\({}_{\text{t}}\)—effect of the interaction of temperature and fiber exposure time, and X\({}_{\text{V}}\)X\({}_{\text{t}}\)—effect of the interaction of propanol volume and fiber exposure time.  
\end{table} 
Table 3: Sources of variation in the ANOVA models for acrylamide extraction (peak area). used (Figure 3). Propanol is immiscible in water forming two phases in the presence of salt [39]. Similar to a solubility modifier effect, the saturation of water with propanol seems to reduce AA solubility, favoring its volatilization to the headspace. Propanol has already been used to AA extraction from food samples [22; 28]. Nevertheless, because AA solubility in propanol is also high [22; 40], when using only propanol, low GC chromatographic areas were observed. Mixture of propanol with water seems to decrease the solubility of AA, resulting in higher AA concentration in the headspace than when these solvents were assayed alone. In the studied samples, water is a solvent serving for AA extraction from solid matrix, while propanol is a solvent promoting AA volatilization in the presence of water. In the case of the fiber exposure time (t) factor, higher AA extractability was achieved when using 15 min of thermostatization followed by 45 min of fiber exposure to the analyte (negative \(b\)-coefficient).