# Volatile Profile of Nuts, Key Odorants and Analytical Methods for Quantification
## 3 Analysis of VOCs Present in Nuts  
The large number of volatile compounds, usually up to 200, that can be identified in nuts, make their identification and quantification difficult [12]. In this case, a separation technique such chromatography is usually employed. The volatile nature of the compounds determined makes gas chromatography (CG) coupled to mass spectrometry (MS) the preferred chromatographic type utilized for this analysis [17]. By using this technique, volatile compounds are commonly introduced in the chromatograph by using the split or splitless mode but if a higher sample capacity is needed due to a low volatile concentration in the sample, a programmed temperature vaporizer (PTV) is employed as  
Figure 3: Scheme showing different pathways from the main precursors of nut volatiles [3; 10]. an injection port [18]. In order to identify the main VOCs, the most extended stationary phases are nonpolar phases such as polydimethylsiloxane, columns with 5% phenyl groups or moderately polar phases such as polyethylene glycols. The length of the employed columns can vary between 25 and 30 m, but if complex mixtures of volatile compounds need to be separated, the length of the column must be increased up to 200 m [18]. Finally, the detector selected is usually a mass spectrometer that allows spectral information of the peaks to be obtained for identification of the target analytes. In routine analysis, the flame ionization detector (FID) can also be employed [18]. Prior to the identification and quantification of volatiles in nuts, the extraction of the target compounds must be optimized. A reference method for the extraction of VOCs in food matrices is liquid extraction (LE) with organic solvents. This methodology implies the employment of great amounts of organic solvents and, in many cases, low recoveries are obtained [19]. To solve this problem, liquid-liquid microextraction (LLME), dispersive liquid-liquid extraction (DLLE), stir bar sorptive extraction (SBSE) and solid-phase microextraction (SPME) are also employed. All of these analytical methodologies are commercially available, and extensively used. SPME is nowadays the most commonly used extraction methodology for the analysis of volatile compounds in food samples, either in direct or in headspace (HS) mode, since it is a solvent-free extraction process that employs lower amounts of sample [17]. The analytical methodology consists of a fiber holder, in which a fiber coated with polymeric material is employed to retain the volatiles. After this, the analytes can be directly desorbed on the injection port of a chromatograph [20]. SPME methodology involves equilibration and extraction steps. Firstly, the sample inside a sealed vial is exposed to a selected temperature for a specific period of time in order to promote analyte volatilization into the HS in the equilibration step. Secondly, the coated fiber is immersed in the HS of the sample, maintaining a constant temperature, for a selected period of time to extract the target compounds. Experimental conditions (time and temperature values) of both steps, equilibration and extraction, commonly employed in VOCs analysis in nut samples are depicted in Table 1.  
SPME results depend on the quantity of sample, and time and temperature extraction values. The influence of both factors--time and temperature--should be analyzed simultaneously since a temperature increase induces a drop in process time in many cases. Consequently, it is reported that higher temperatures reduce the needed equilibration time [21]. However, shorter equilibration times produced higher relative standard deviations (RSD) [19]. [PERSON] et al. tested higher temperatures (50, 60, 70 and 80 \({}^{\circ}\)C) and short equilibration times (5-60 min) [15]. Optimal values (60 \({}^{\circ}\)C, 10 min) proved a remarkable time reduction at higher temperatures. In addition, ultrasonic extraction has been used in place of the extraction by using temperature for analyzing the volatile compounds present in almonds [22].