## 2 Tools for Sampling Semiochemicals to Study Insect Interactions
### Most Commonly Used Methods for Extracting Sentiochemicals
#### 2.1.1 Solvent-Assisted Extraction (SAE)  
This method is one of the most widely used for VOCs and other metabolites obtained from biological sources in general due to its easy reproduction, versatility in implementation and relatively low cost. The technique consists of contact of a solvent with the biological sample for which extraction will be performed (which can be a whole organism or a portion of the same, such as an organ or tissue). Such a solvent penetrates the matrix and elutes from it with a variable quantity of dissolved metabolites, depending on the involved solubility characteristics. Thus, the metabolite-enriched solvent mixture is then separated from the original matrix affording the crude extract.  
The most basic version of SAE is the common solid-liquid extraction, which in turn is the most utilized of the SAE methods for VOC extraction [21; 22; 23; 24; 25; 26; 27; 28; 29; 30]. The extraction effectiveness is variable according to the sample itself and the correct selection of the solvent, and for VOCs, the low polarity organic solvents such as hexane, pentane and dichloromethane are preferred. The results can be enhanced by manipulating physical parameters such as mass transfer (stirring), temperature [3], and surface. A classic example of exploitation of these parameters is Soxhlet extraction, which achieves continuous sample extraction by solvent recycling derived from successive in-situ distillations, allowing the solvent to enrich on compounds from the sample with increasing solubility derived from heating.  
Instrumental tools are also available for increasing the SAE efficiency, which manipulate the physicochemical properties of the solvent in a systematic manner. One of these approaches is microwave-assisted solvent extraction (MASE) [3], in which radiation is employed for quick heating of the sample and shortening of the process duration. For MASE, the use of a solvent that absorbs microwave radiation is necessary. One of these other techniques is supercritical fluid extraction, especially with carbon dioxide (CO\({}_{2}\)) for VOC collection [25]. The SFE exploits the attributes of CO\({}_{2}\) as a supercritical fluid to obtain the best solvent qualities compared to the regular organic solvents, such as greater sample penetration, and once the process has ended, the CO\({}_{2}\) can be reverted to its gas phase, facilitating its removal by evaporation, with no residues in the sample. Finally, in ultrasound-assisted extraction [25; 31], a sound frequency is applied to the material that is being extracted to promote its fragmentation and poration at the cellular scale, then the compounds diffuse more rapidly to the solvent. This is performed at low temperatures to minimize thermolabile compound loss, but isomerization or decomposition of chemically unstable compounds can take place [3].  
Some complementary treatments for SAE can be applied with the purpose of pre-concentrating the existing VOCs from the extract prior to the analysis when the concentration of the metabolite of interest is lower than required. For example, a stirring-bar sorptive extraction (SBSE) can be performed on solvents with low VOC concentrations and then the volatile compounds can be recovered using a more affined solvent with successive washing/storing cycles. This method has been shown to be effective in the concentration of VOCs recovered from water and involved in the relationship between plants of _Potamogeton perfoliatus_ and _Myriophyllum spicatum_ and the aquatic beetle _Macroplea appendiculata_; the concentration of the VOCs in the original extract would be problematic for analysis and difficult to recover using other techniques [26]. Additionally, solvent-assisted flavor evaporation (SAFE) is a vacuum distillation that allows VOCs to separate from nonvolatile substances in the original extract, with successful utilization in the preconcentration of VOCs from insects [30].