## 3 Results and Discussion  
### Optimization of HS-SPME Parameters  
Milled rice, of the variety Pusa Basmati-1121, was used for the optimization of HS-SPME conditions. Initially, the extraction performance of volatile compounds in rice sample was tested using three different SPME fibers, such as CAR/PDMS, PDMS/DVB, and 50/30 \(\upmu\)m DVB/CAR/PDMS, by comparing the abundance of volatile compounds in the respective total ion chromatograms (Figure S2). This study showed that 50/30 \(\upmu\)m DVB/CAR/PDMS fiber extracted the maximum number of volatile compounds, as found in the previous studies [9; 14]. In subsequent steps, using 50/30 \(\upmu\)m DVB/CAR/PDMS fiber, the optimal incubation temperature and extraction time were estimated to extract the maximum amount of 2-AP from the raw rice sample at three different sample weights. Figure S3 shows the results of the optimization of HS-SPME conditions for the extraction of 2-AP at different incubation temperatures, extraction times, and sample weights. The highest recovery of 2-AP at the shorter extraction time and minimal incubation temperature was the main criteria for determining the optimal extraction conditions. In this study, all the studied parameters, such as incubation temperature, extraction time, and sample weight, were found to have a considerable influence on the extraction yield of 2-AP. An increase in the 2-AP content was observed with an increase in incubation temperature and extraction time up to 60 \({}^{\circ}\)C and 15 min, respectively. Higher incubation temperature and extraction time above 60 \({}^{\circ}\)C and 15 min, respectively, led to either no change or a decrease in the 2-AP level. Similarly, at the 1.0 g sample weight, the extraction yield of 2-AP was maximum. These findings, altogether, showed that one gram of sample incubated at 60 \({}^{\circ}\)C for 15 min was the optimal HS-SPME condition to extract the amount of 2-AP from the raw rice sample. Previous studies also have shown 60 \({}^{\circ}\)C as the optimal incubation temperature for the extraction of 2-AP and other volatiles [16; 17]. In several other studies, 2-AP from rice samples was extracted at a higher incubation temperature, \(\leq\)80 \({}^{\circ}\)C [6; 18; 19]. However, as seen in this study, the extraction temperature over 75 \({}^{\circ}\)C was not found to be suitable to obtain an optimal yield of 2-AP; this is possibly because of a loss of volatiles, including 2-AP, occurring at high extraction temperature may be due to the failure of the vial cap septum [9; 19]. As per the literature, an equilibration time of 10-15 min is generally sufficient to extract the majority of volatile compounds. Akin to the findings of this study, an extraction time of 15 min was found to be optimal to achieve a maximum yield of 2-AP from rice [20].  
In previous studies, rice samples were cooked in the headspace vial by adding water [8; 20]. However, the amount of water added in proportion to the weight of the rice sample was found to be crucial to obtain a maximum yield of 2-AP from cooked rice by HS-SPME [20]. Considering this, the amount of water required for the optimal extraction of 2-AP from a cooked rice sample was optimized at 60 \({}^{\circ}\)C for 15 min incubation in a subsequent study. Compared to parts of water (mL) to rice (g) ratios of 0.5:1.0, 1.0:1.0, and 2.0:1.0, a smaller amount of water (0.250 mL) added to the rice (1.0 g) considerably increased the amount of 2-AP (Figure S3b). The addition of water over 0.25 mL into the one-gram rice sample containing vial was found to remarkably reduce the extraction yield of 2-AP. Taken together, the incubation of a 20 mL vial containing one gram of rice grain sample and 0.25 mL of water at 60 \({}^{\circ}\)C for 15 min was the optimal HS-SPME experimental condition for the extraction of 2-AP from cooked rice.