# Examination of Intermolecular Forces Influencing Headspace Analysis of Biological Samples
## 4 Discussion  
Chemical analysis of volatiles from biological samples can provide useful insights into metabolic processes and may serve as biomarkers to monitor diseases or health status. Headspace analysis has a distinct advantage of minimal sample preparation for this purpose. However, quantitative analysis relies upon the efficient transfer of volatiles from the sample into the gas chromatograph. Not only are headspace sampling approaches inefficient for compounds with low vapor pressure, but headspace sampling can also be poorly quantitative because volatile collection is subject to multiple equilibria. Thus, without correction, concentrations observed in the headspace may not accurately reflect concentrations present in the sample. This study represents the effort to demonstrate sample matrix effects on headspace concentrations of volatiles and to overcome this to achieve more accurate analyses. Significant reductions in the headspace responses of volatiles with higher K\({}_{\text{ow}}\) values were observed when the sample matrix was less polar. For example, the headspace concentrations of 2-nonanone (K\({}_{\text{ow}}\) = 3.16) were significantly lower in intralipid versus water (Figure 1). In general, headspace concentrations of all volatiles from serum were reduced in comparison to water. These differences could be mediated by normalization to the internal standard in some cases. However, while acetophenone-2\({}^{\prime}\),3\({}^{\prime}\),4\({}^{\prime}\),5\({}^{\prime}\),6\({}^{\prime}\)-d5 accounted for solubility differences in octanal, it failed to normalize the 2-nonanone response in the lipid sample due to its great affinity for lipids. Therefore, in some cases, multiple internal standards might be required for improving quantification in analyses [31]. While salting-out may enhance the efficiency of headspace analysis, its effect also varies between compounds and it does not eliminate matrix effects, potentially causing inaccurate results when dealing with highly complex matrices or analytes with similar properties to matrix [32, 33]. Secondly, large reductions in headspace concentrations may result from the strong interaction between the volatile molecules and other solutes in the sample matrix. It was found that headspace concentrations of aldehydes were significantly reduced from the serum sample matrix (Figure 2). Aldehydes, produced by a lipid peroxidation, easily react with proteins in blood via a wide variety of intra- and inter-molecular covalent adducts. They can form Schiff bases and/or Michael adducts with the free amine group of lysine, the imidazole group of histidine, the guanidine group of arginine, and thiol group of cysteine [34]. This interaction can significantly alter aldehyde properties, affecting extraction \begin{table}
\begin{tabular}{c c c c} \hline
**Volatile** & **a-Term** & **b-Term** & **Intercept (C)** \\ \hline
1-Octanol & 0.3676 & 0.1251 & 0.0101 \\ Octanal & 0.0033 & 0.0012 & 0.0009 \\
2-Octenal & \textless{}0.0001 & \textless{}0.0001 & 0.0001 \\
2,4-Octadienal & \textless{}0.0001 & \textless{}0.0001 & \textless{}0.0001 \\ Acetophenone-2\({}^{\prime}\),3\({}^{\prime}\),4\({}^{\prime}\),5\({}^{\prime}\),6\({}^{\prime}\)-d5 & 0.2471 & 0.1535 & 0.0749 \\ \hline \end{tabular}
\end{table} 
Table 2: ANOVA probabilities for analysis of media effects (serum, intralipid, or water) on the polynomial terms describing the relationship between incubation/extraction temperature and headspace concentration. The second-order function follows the form Y = aX\({}^{2}\) + bX + C.