# Application of Thin-Film Microextraction to Analyze Volatile Metabolites in A549 Cancer Cells
## 2 Results and Discussion
### Optimization of the TFME Extraction Procedure
Nonetheless, quick achievement of a new equilibrium in a relatively small, closed system (5 mL liquid medium in 20 mL vial) and fast extraction kinetics on TFME mesh, along with no restriction to VOCs diffusion from the headspace to the sorbent (in contrast to deteriorated transfer through glass wool into a TD tube), resulted in higher extraction rates for IN-HS compared to EX-HS. Although an ambient extraction temperature of \(\sim\)20 \({}^{\circ}\)C is very convenient, it has important drawbacks, namely it is not optimal for cultured cell lines and microorganisms, and it may be unstable within an intra and interday period. Therefore, a temperature of 37 \({}^{\circ}\)C for TFME extraction was also studied using a sampling protocol elucidated from previous measurements, i.e., HLB material and IN-HS mode. According to the obtained results, an elevated temperature improved extraction efficiency for all VOCs detected (Figure 3). This is a consequence of enhanced vaporization of volatile compounds from the aqueous medium, hence their faster distribution within the headspace gas and better penetration of the sorbent solid phase. Simultaneously, the temperature of 37 \({}^{\circ}\)C seems to be sufficiently low to avoid undesired depletion in the exothermic adsorption process. In the search for improved sensitivity, so much needed in ultra-trace analysis, the extraction phase volume is often enlarged. Despite the obvious benefit of increased capacity for the analyte, this approach results in a prolonged equilibration time and hinders desorption efficiency. Therefore, in TFME, the geometry of the device is designed to ensure an effectively high sorbent area-to-volume ratio by deposition of an extraction phase on the flat planar mesh. [PERSON] et al. (2018) demonstrated that this process was crucial to decrease the background from the TFME device itself, whereby high-density PDMS prepolymer was used as a thermally stable glue to support the extraction phase on a carbon (instead of fibreglass) mesh yielding minimal bleeding of siloxanes. Given the importance of the extraction time in SPME sampling, the following durations were tested: 10, 20, 35, 60 and 90 min. According to the results gathered, the higher the molecular weight (and lower volatility), the longer time needed (Figure 4). This is evident for aldehydes and particularly for alcohols, where the initially weak influence of extraction time on amounts of adsorbed small molecules within those groups increased for heavier compounds. A similar relationship was maintained over the full range of hydrocarbons investigated here. In turn, the amounts of extracted compounds containing sulfur (e.g., methanethiol, CS2) or nitrogen (e.g., pyrrole, methylpyrazine) were inversely proportional to extraction time, suggesting competition and perhaps displacement of these molecules from the HLB sorbent with analytes containing more electronegative oxygen in their molecules. Due to practical reasons, extraction times longer than 90 min were not studied. Figure 2: Optimization of sampling protocol for VOCs extraction from a pooled medium headspace. The \(y\)-axis represents log-transformed peak area values, where means are given with a respective standard deviation for three replicates (\(n=3\)) for each sampling mode: external headspace (EX-HS, grey boxes) and internal headspace (IN-HS, blue boxes). Figure 3: Influence of temperature on VOCs extraction from a pooled medium headspace on HLB sheets according to IN-HS sampling protocol. The \(y\)-axis represents log-transformed peak area values, where means are given with a respective standard deviation for three replicates (\(n=3\)) for each temperature: ambient of \(\sim\)20 \({}^{\circ}\)C (“cold”, grey boxes) and elevated to 37 \({}^{\circ}\)C (“heated”, blue boxes). Figure 4: Optimization of extraction time.