## 3 Results and Discussion
### Method Evaluation  
To evaluate the efficacy of the method optimization, we conducted a comparison of the optimized and pre-optimization methods in terms of total peak area and total peak number. The chemical class composition of the extracted compound is important as it may be indicative of the origin of the volatile compounds. Therefore, we examined the peak area and peak number in nine chemical classes. In addition, we examined the number of compounds consistently present in all mixed BALF samples to evaluate if the optimized method can decrease the number of missing values caused by the sample extraction step. We hypothesized that the optimized HS-SPME method would outperform the pre-optimization method regarding total peak area and peak number. We observed that optimizing the method led to increased extraction efficiency for all 10 pairs of mixed BALF samples. Specifically, the optimized method generated an average total peak area of \(4.98\times 10^{7}\), a 340% increase compared to the pre-optimization method, which was statistically significant (\(p\)-value < 0.0001) (Figure 7a). Additionally, the optimized method generated an average of 80% more peaks compared to the pre-optimization method, also showing a statistically significant difference (Figure 7b). Our findings show that the optimization of vial size, dilution factor, extraction time, extraction temperature, and salt concentration improves the HS-SPME efficiency regarding total peak area and total peak number. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Optimized Parameter** & **Pre-Optimization** & **Optimized** \\ \hline Vial Size & 20 mL & 10 mL \\ Dilution Factor & 1:2 Dilution & No Dilution \\ Extraction Time & 30 Minutes & 50 Minutes \\ Extraction Temperature & 43 \({}^{\circ}\)C & 45 \({}^{\circ}\)C \\ Salt Addition & No Salt & 40\% (\(w/v\)) NaCl \\ \hline \hline \end{tabular}
\end{table} 
Table 2: HS-SPME parameters for the pre-optimization and optimized method. Figure 7: (**a,b**) Comparison of the average total peak area and total peak number of 10 sets. (**c,d**) Illustration of the variation in the peak area and peak numbers among nine chemical classes. *** \(p\)-value < 0.0001. We also compared the peak area and peak number for nine major chemical classes in the BALF samples extracted using the pre-optimization and optimized methods. We observed that, after optimization, the total peak area increased for nine major chemical classes (Figure 7c). The increase ranged from 112% to 761%, among which alcohols, ketones, and esters have the most significant increase. Specifically, the alcohols increased by 761%, on average, compared to the pre-optimization method; the ketones increased by 447%; and the esters increased by 425%. All nine classes demonstrated statistically significant differences except the aliphatic hydrocarbons. The total peak number also increased for all nine chemical classes (Figure 7d), with the increase ranging from 19% to 116%. The alcohols, halogenated compounds, and esters showed the most notable increase of 116%, 103%, and 79%, respectively. All nine classes demonstrated statistically significant differences. We noticed that the chemical classes with polar functional groups demonstrated a more significant increase. Thus, we may speculate that optimization has a more significant impact on polar compounds. In addition, we hypothesized that the optimized method would increase the number of extracted volatile compounds consistently present in BALF samples. The pre-optimization samples had 56 features consistently detected in samples, while the optimized method had 108 features consistently detected (Figure 8). We conclude that increased peak intensity can make the peak identification more consistent. Among the 108 identified compounds, 52 compounds were presented in both methods. Fifty-six compounds were only identified with the optimized method. Only four compounds that were constantly present using the pre-optimization method were not observed in the optimized method.