## 3 Results and Discussion
### HS-SPME Method Optimization
#### 3.1.4 The Effect of Salt Addition  
We hypothesized that adding salt to the sample would lead to a higher total peak area and total peak numbers. The results show that adding 40% NaCl to the samples significantly enhanced the number of extracted peaks and total peak area, compared to 20% and no salt addition (Figure 4a,b). Specifically, the samples with 40% salt obtained an average total peak area count of 3.44 \(\times\) 10\({}^{7}\), whereas samples with 20% salt and samples without salt obtained 3.08 \(\times\) 10\({}^{7}\) and 2.05 \(\times\) 10\({}^{7}\), respectively. The samples with 40% salt obtained an average total peak number of 373, while those with 20% and no salt obtained an average total peak number of 358 and 312, respectively. For both the total peak area and total peak number, the difference between 40% and no salt was statistically significant. Figure 3: Response surface analysis performed on total peak area and total peak number. (**a**) Total peak area vs. extraction time vs. extraction temperature. (**b**) Total peak number vs. extraction time vs. extraction temperature. Yellow dots represent the optimum point. Salt addition is a common technique to enhance HS-SPME efficiency. Adding salt may increase the activity coefficient of the compounds in the sample, which subsequently leads to an increase in the concentration of the compounds in the headspace [3]. The effect of salt addition depends on salt concentration [53], and it is essential to experimentally evaluate and optimize this parameter. Our finding aligns with other studies, which reported that adding NaCl enhanced HS-SPME extraction efficiency in various sample types, including urine [60], culture media [48], water [61], liquor [56], and cow slurry [62]. We observed that although the average total peak area and peak number were higher in samples with 40% NaCl than those with 20%, the difference was not statistically significant. Similar findings have reported that the salt addition effect is non-linear and tends to reach a plateau at 35% NaCl, approaching the saturation concentration of 40% [32]. It has been mentioned in the theory of HS-SPME that the salt addition effect increases with increasing compound polarity [53]. Therefore, we also hypothesized that the salt addition would affect polar compounds more than non-polar compounds. To test this hypothesis, we evaluated the salt addition effect on 46 compounds (Supplementary Materials, Table S2), which were selected based on the criteria mentioned in Section 3.1.3. The correlation analysis between the partition coefficient \(K_{\omega w}\) (octanol/water partition coefficient, used as a substitute for polarity) and the Log2 fold change of each compound showed a negative regression trend (Figure 5). The slope is significantly different from zero (\(p\)-value \(<\) 0.001). Furthermore, we selected the two most polar (2,3-butanedione and ethanol) and the two most non-polar compounds (decane and 1,3-bis (1,1-dimethyl ethyl)-benzene). We studied their variance of relative abundance with increased salt amount. The results showed that for 2,3-butanedione and ethanol, the peak area differences among three NaCl concentration levels were statistically significant, except for 2,3-butanedione between 20% and 40% NaCl. For the two non-polar compounds, while decane exhibited no noticeable variation in peak area across different NaCl concentrations, 1,3-bis(1,1-dimethylethyl)-benzene displayed a decrease with increasing salt concentration (Figure 6). Based on our observation, we can conclude that adding salt can significantly improve the HS-SPME efficacy. We can also speculate that the salt addition predominantly affects the polar compound rather than the non-polar compound, and the extent of the salting addition effect might correlate with the polarity of the compounds. Figure 4: (**a**,**b**) Comparison of total peak area and total peak number of peaks extracted among samples with no salt, 20% salt, and 40% salt; * \(p\)-value \(<\) 0.05; **\(p\)-value \(<\) 0.01; *** \(p\)-value \(<\) 0.001.