# Examination of Intermolecular Forces Influencing Headspace Analysis of Biological Samples
## 4 Discussion
\begin{table}
\begin{tabular}{c c c c c} \hline
**Volatile** & **Log K\({}_{\text{ow}}\)** & **Serum** & **Intralipid** & **Water** \\ \hline
1-Octanol & N/A & 69.7 \({}^{\circ}\)C & 68.2 \({}^{\circ}\)C & 59.6 \({}^{\circ}\)C \\ Octanal & 2.55 & 63.5 \({}^{\circ}\)C & 40.5 \({}^{\circ}\)C & 41.4 \({}^{\circ}\)C \\
2-Octenal & 2.57 & 67.6 \({}^{\circ}\)C & 61.1 \({}^{\circ}\)C & 39.0 \({}^{\circ}\)C \\
2,4-Octadienal & 2.35 & 70.7 \({}^{\circ}\)C & 68.7 \({}^{\circ}\)C & 64.1 \({}^{\circ}\)C \\ Acetophenone-2\({}^{\prime}\),3\({}^{\prime}\),4\({}^{\prime}\),5\({}^{\prime}\),6\({}^{\prime}\)-d5 & 1.65 & 68.5 \({}^{\circ}\)C & 68.2 \({}^{\circ}\)C & 65.3 \({}^{\circ}\)C \\ \hline \end{tabular}
\end{table} 
Table 3: Critical values (incubation/extraction temperature associated with maximum headspace concentration) for each volatile in serum, intralipid, and water media. efficiency for these important metabolites. Relative to water, the headspace concentration of the aldehydes in other matrices increased when proteins were removed (Figure 2). Similarly, the addition of a protein (BSA) reduced the headspace concentration of several aldehydes due to these same intermolecular forces (Figure 3). Together, these results revealed that the proteins were the main source of the interactions with the volatiles and resulted in reduction in headspace concentration. Although a significant number of metabolite-protein interactions have already been discovered, only a small portion of interaction networks have been identified [35]. Unraveling the interaction networks by systematic approach can further help to understand the molecular basis of healthy and diseased states and promote the availability of unbound volatile metabolites in headspace analyses. One way of improving the sensitivity of the headspace analysis is to raise the temperature of the sample to drive the equilibrium closer to the vapor phase [36]. It was also anticipated that the interactive forces between volatiles and media might become negligible at higher temperatures. Elevated temperatures from 40 to 100 \({}^{\circ}\)C were evaluated (Figure 4). Critical values (temperature associated with highest headspace concentration) widely varied in water (Table 3) and were elevated in serum. Even though the factors controlling equilibrium in headspace analyses may be very complex, an incubation and extraction temperature of 70 \({}^{\circ}\)C appears to be the optimal extraction temperature for volatile metabolites in biological samples. In conclusion, the volatile amounts detected in the headspace from the samples were notably affected by the sample matrix, and the deviations were determined not only sample matrix, but also structural properties of the volatile molecules. The solubility of an analyte in the matrix played a key role in determining its mass transfer into the headspace. The greater the solubility, the less likely the analyte was to partition into the headspace, resulting in lower headspace responses by GC-MS. A significant reduction in headspace concentration was noted for aldehydes, more specifically conjugated aldehydes. One challenge in quantifying reactive aldehydes in biosamples would be the possible requirement of additional sample processing [37]. The matrix effects may be overcome by increasing the extraction temperature to inactivate blood proteins in a HS-SPME procedure. The optimized extraction temperature of 70 \({}^{\circ}\)C benefitted recovery of these important oxygenated volatiles. Overall, these results further demonstrate the need for proper method development and validation. It is essential to emphasize that no one set of conditions can be considered sufficient for broad applications in biomarker discovery.