## 3 Results & Discussion
### HS-SPME-GC-MS--Headspace Solid-Phase Microextraction Coupled to Gas Chromatography
#### 3.3.2 Validation method  
The analytical method used for the quantification of volatile compounds by HS-SPME-GC-MS was validated in terms of linearity, repeatability and detection (LOD) and quantitation (LOQ) limits. An acceptable level of linearity was obtained for all analytes (R\({}^{2}\) between 0.9076 and 0.9929), showing relative standard deviation (RSD) values lower than 5%. LOD and LOQ values were determined using the regression parameters of the calibration curves (3 Sy/x/a and 10 Sy/x/a, respectively, where Sy/x is the standard deviation of the residues and \"a\" is the curve slope). LOD and LOQ values obtained for heptanal ranged between 0.001 and 0.004 ug/kg, \(\alpha\)-methylstyrene ranged between 2.76 and 9.19 ug/kg, benzaldehyde ranged between 0.003 and 0.011 ug/kg, octanal ranged between 0.071 and 0.236 ug/kg, 1-hexanol and decanal ranged between 0.001 and 0.002 ug/kg. Figure 4: Pareto charts of factors and interactions obtained from the BBD for the sum of the volatile compounds. The vertical line indicates the statistical significance at 5% of the effects. Volatile Compounds Quantification  
Lipid oxidation is a complex process where unsaturated fatty acids react with molecular oxygen via a free radical mechanism or in a photosensised oxidation process. The principal source of off-flavours developed by lipid oxidation is hydroperoxides, which are unstable and readily decompose to form aliphatic aldehydes, ketones and alcohols. Many of these secondary oxidation products have undesirable odours with particularly low odour thresholds [53; 54]. As it is shown in Tables 3 and 4, the effect of studied nanoclay (T1, T2 and T3) were different depending on the polymer matrix (PHB226, PHA1005 or PHA3002). In general, the volatile compound quantified in greater quantity for all the samples has been the decanal as it is the volatile with the highest \(M_{\mathrm{w}}\). Table 4 results suggest that there is a clear tendency which indicates that the content of the volatiles is related to the molecular weight, as the higher the \(M_{\mathrm{w}}\) is, the more difficult the volatiles are to extract from the polymer and to clean during purification stages. Therefore, we can observe them in higher amounts. For the case of \(\alpha\)-methylstyrene and benzaldehyde, these are more complex volatiles containing aromatic groups which may be also less accessible to solvents to be extracted and cleaned. Hence, they can be found also in higher quantities than simpler alcohols. According to [PERSON] et al. [30], the addition of nanoclays may modify the structure of the polymers studied affecting the release of the studied undesirable volatile compounds. Regarding the control matrix polymers, PHB 226 initially shows lower contents of heptanal, \(\alpha\)-methylstyrene, octanal and 1-hexanol while its content in decanal was the highest respect to the other two PHA matrices. However, benzaldehyde was initially quantified in similar amounts in all control matrices. Concerning the effect of the addition of T1, T2 and T3, each studied volatile showed a different behaviour depending on the matrix and organolay used (Figure 5 and Table 5). Regarding 1-hexanol, PHB 3002 showed the lowest content in contrast to PHA1005 and PHB226. In this sample, no significant enhancement was found between the three nanoclays, although T1 at least maintained the level of release, while T3 and T2 showed non-desirable and remarkable increasing peaks of 200% and 515% respectively. For PHA 1005 all the nanoclays reduced the release of this compound, but it seems that T3 clay was much more effective reducing it in a 72%. Heptanal, together with Decanal, seemed to be the most difficult volatile to be adsorbed by any of the organoclays. In this sense, T1 seemed to be more efficient maintaining heptanal release for PHA1005 and PHA3002 and reducing a 20% the release PHB226. In contrast, it seems that the addition of T3 could modify the structure of PHA1005 and PHA3002 polymers increasing the heptanal release in more than 400% while reducing 13% for PHB226.