## 3 Results and Discussion
### Effect of Extraction Solvents  
Figure 3 shows the hierarchical cluster analysis (heatmap) in the form of a dendrogram. The heatmap indicates the relative intensity of four biological replicates which depended on the peak area of each compound in each solvent. Chloroform is not included in the figure because the SPME fiber coating was dissolved in this solvent. The highest relative GC response of the compound in comparison to the other was specified a \"hot\" color, while those that are lower in their values were given a \"cold\" color. The top of the dendrogram indicates the similarity among the solvents and the data variation among the replicates (Figure 3). The side arrows show a major compound with high area GC response in each solvent (Figure 3). The compounds obtained from the eight solvents consisted of an assortment of fatty acids, hydrocarbon waxes, and sterols in addition to some metabolic products, such as p-benzoquinone and methyl-p-benzoquinone (Supplementary Material Table S1). Fifty-three compounds were acquired from a total of eight tested solvents. Acetonitrile and the combination of acetonitrile and ethanol had the highest peak numbers with 41 and 34 compounds, respectively,  
including most of the fatty acids and hydrocarbon waves, while the lowest number of compounds (22) (Table 1) was obtained from acetonitrile and water. It was observed that each compound had a different GC response according to the solvent used in the extraction process. Both acetonitrile and the combination of acetonitrile and ethanol showed a similar influence regarding the GC response of compounds. Most of the highest molecular weight compounds had higher intensities when acetonitrile and the combination of acetonitrile and ethanol were used as solvents. On the other hand, both the combination of acetonitrile and ethanol and water and the combination of acetonitrile and water had parallel effects (Figure 3). This could have been because of the similar polarity of the combined solvents. However, a higher GC response of some main fatty acids was detected in the combination of acetonitrile and water, such as palmitic acid (23.37_135), stearic acid (26.42_143) and linolenic acid (26.05_137) (Figure 3 and Supplementary Material Table S1) because these are more polar compounds and the presence of water in a solvent increase the polarity of the solvent. While the sterol compounds showed a higher GC response under the acetonitrile and ethanol and water solvent, methanol and ethanol were quite similar in their extraction abilities, although methanol efficiently extracted most of the high boiling point hydrocarbon waves. However, the use of methanol as an extraction solvent may form artificial methyl esters of fatty acids in the presence of some organic or inorganic materials [30]. As per Figure 3, with hexane, most of the compounds were between retention time (RT) = 6.65 to 15.55 min and not at higher RT where C25-C34 compounds for _Tribolium_ and _Rhyzpertha_ could be seen, though hexane was commonly used as a solvent to extract the cuticular lipids from many insects [13; 31]. However, a low yield of hydrocarbon waves in this study could have be due to the fact of the incompatibility of fiber coating and the solvent as a result a lack of the efficient distribution of analytes between the fiber and the solvent. Chloroform is another solvent which has been successfully used as a single solvent [32] or combined with another solvent such as hexane [16] to extract insect lipids, producing a high yield [1]. However, this was not appropriate to use for the DI-SPME technique as it can destroy the SPME fiber coating. Figure 1: Total signal chromatogram of lipids of cuticular extraction followed by homogenized body extraction of same insects of _Tribolium castaneum_, using acetonitrile as solvent. From the above results, acetonitrile was selected as the optimal solvent in the validation study.