# Volatile Profile of Nuts, Key Odorants and Analytical Methods for Quantification
## 3 Analysis of VOCs Present in Nuts
Meanwhile, the sample is often agitated using a stirring bar to decrease the time necessary for equilibration [23]. However, as is shown in Table 1, a steady extraction process is commonly used as well. In fact, [PERSON] et al. demonstrated that stirring has a minimal effect in the extraction yield of highly volatile compounds [24]. In addition, the solid nature of nut samples makes their agitation difficult, and the addition of water is necessary for homogenization purposes [22]. Moreover, some researchers increased the medium ionic strength in order to modify the nature of the matrix and the extraction efficiency, since this addition could affect the partition coefficient of the analytes [25,26]. Nevertheless, the higher the ionic strength, the lower the solubility of neutral molecules in water and the less likely these molecules are to pass from the solid matrix to the water. As a result, the extraction efficiency of these molecules decreases [19]. Consequently, most authors decided to avoid the addition of salt [19,27,28,29]. It is interesting to highlight that no water addition was required when hexanal was the only target compound [15]. Sample amount is also a key factor in the extraction process of volatile compounds in nuts [15]. As can be seen in Table 1, different sample amounts have been tested, most of them being higher than 0.1 g.  
Finally, analyte extraction depends on fiber type. The commercially available fiber materials are carboxen (CAR), polydimethylsiloxane (PDMS) and divinylbenzene (DVB) [21]. In order to choose the most suitable extraction phase composition, the highest affinity of the target analytes is the key factor in the extraction efficiency. The vast majority of authors opt to employ three-phase CAR/PDMS/DVB fiber because of its high extraction capacity, which allows the removal of a high number of odorants [19]. Nevertheless, depending on the target compound, other coatings could also be better for the extraction process. As an example, [PERSON] et al. showed that CAR/PDMS fiber had higher sensitivity for hexanal extraction than PDMS, PDMS/DVB, CAR/DVB, and DVB/PDMS/CAR fibers [15]. Once the results are obtained, some authors directly employ the area of the compound [19; 30; 31] (or the percentage of areas [32; 33; 34]) if the analysis has the aim of comparing different varieties or the processing conditions of nuts. However, in some studies, a complete quantification was performed. Different types of quantification approaches are shown in Table 1. Most researchers employ semi-quantification with internal standard (IS), which is described in Equation (1), Equation (2) being an extension of the previous one. In both cases, the procedure is simple due to the fact that an external standard (ext. std.) curve is not required. Actual quantification is depicted in Equation (3). External standards are commonly prepared in deionizer water [35] or in organic solvents (e.g., n-hexane [25]). \[\text{Concentration}=\frac{\text{Extraction ion peak area}}{\text{Extracted ion peak area of IS}}[\text{IS}] \tag{1}\]  
\[\text{Concentration}=\frac{\text{(Extracted ion peak area)}/\text{( Extracted ion peak area of IS)}}{\text{Sample weight}}[\text{IS}] \tag{2}\]  
\[\text{Concentration}=\frac{\text{Extracted ion peak area}}{\text{Slope of Ext.Std.curve}} \tag{3}\]  
[PERSON] et al. prepared external standards in a devolatized nut matrix, adding a mix solution of deuterated analytes [12; 21]. Deuterated compounds were employed as internal standards. Octanal-d16, 2-methylpyrazine-d6, and n-hexyl-d13 alcohol were used as internal standards to determine aldehydes, pyrazines, and alcohols, respectively [36; 37; 38]. Octanal, 2-methylpyrazine and 1-hexanol were also employed for this purpose [22]. However, other studies used only a compound as the internal standard such as 4-methyl-2-pentanone [2; 39] or 2-pentanol in pistachios [28]. Additionally, it is important to keep in mind that there are great differences in the perception threshold concentrations in humans for the different volatile compounds found in nuts.