## 2 Results and Discussion
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \multirow{2}{*}{**Compounds**} & \multirow{2}{*}{**RI 1a**} & \multirow{2}{*}{**RT b**} & \multicolumn{4}{c}{**Mating Stages**} & \multirow{2}{*}{\(p\)**Value**} & \multirow{2}{*}{**FDR 4**} \\ \cline{3-3} \cline{5-8}  & & & & **Before** & & **During** & **After** \\ \hline Dodecanoic acid & 1572.6 & 17.342 & N.D c & N.D c & 104.884 & 0.003 & 0.015 \\
1-Iodododecane & 1716.2 & 19.656 & N.D c & N.D c & 108.690 & 0.002 & 0.014 \\ Tetracosaene & 2078.5 & 25.429 & 110.994 & N.D c & N.D c & 0.001 & 0.014 \\ _trans_-13-Octadecenoic acid & 2122.7 & 26.132 & 361.845 & 980.758 & N.D c & 0.002 & 0.014 \\ (Z)-Oleic acid & 2130.2 & 26.249 & N.D c & N.D c & 618.081 & 6.670 & 0.014 \\ Octadecanoic acid & 2142.1 & 26.434 & N.D c & N.D c & 209.611 & 0.005 & 0.018 \\
9-(Z)-Tricosene & 2244.1 & 28.066 & N.D c & N.D c & 211.876 & 0.001 & 0.014 \\ Hexadecane & 2268.5 & 28.452 & 96.985 & N.D c & N.D c & 0.002 & 0.014 \\
1-Eicosanol, TBDMS derivative & 2327.8 & 30.144 & 44.947 & N.D c & N.D c & 0.003 & 0.015 \\ Supreme & 2748.8 & 36.122 & 434.511 & N.D c & N.D c & 0.007 & 0.024 \\
2-Methyloctadecosane & 22785.6 & 36.698 & N.D c & 44.210 & N.D c & 0.003 & 0.015 \\ Dieethylcycloxyborane & 2831.5 & 37.430 & 66.238 & N.D c & N.D c & 0.001 & 0.014 \\
3,5-Cyclo-6,814,22-ergostatriene & 2873.7 & 38.086 & 64.498 & N.D c & N.D c & 7.440 & 0.014 \\ Hentriacontane & 2969.3 & 39.616 & N.D c & N.D c & 0.03452 & 0.009 & 0.024 \\ Octatriacontrol pentafluoropropionate & 2991.1 & 39.964 & N.D c & 0.002 & 70.866 & 0.004 & 0.015 \\
1,54-Dibromotetrapentacontane & 3017.3 & 40.379 & N.D c & 0.002 & 72.014 & 0.002 & 0.014 \\
9-(Z)-Hexadecenoic acid hexadecyl ester & 3131.3 & 42.196 & 55.305 & 214.519 & 1583.587 & 9.960 & 0.014 \\
11,13-Dimethyl-12-tetradecen-1-acetate & 3137.0 & 42.888 & N.D c & N.D c & 139.731 & 0.003 & 0.015 \\
9-(Z)-Coctadecenoic acid & 3521.9 & 44.119 & N.D c & 76.6686 & 600.066 & 0.002 & 0.014 \\ \hline \end{tabular}  
* RI id retention index; b RT is retention times; c ND is not detected; d FDR is false discovery rate of data. Each number represent the mean of three biological replicates.  
\end{table} 
Table 1: Significant compounds peak area (one unit corresponds to a 10\({}^{4}\) area) detected at three mating stages of medfly by DI-SPME-GC-MS in acetonitrile/water solvent. For some compounds, there were significant differences observed between samples collected at different stages. In the first solvent extraction, tetracosane; diethyldecyloxyborane, 9-(_Z_)-tricosene, hexacosane; 9-(_E_)-octadecenoic acid, 1,54-dibromotetrapentacontane, _trans_-13-octadecenoic acid, 2-methyloctacosane, 11,13-dimethyl-12-tetradecen-1-ol acetate; TBDMS-1-eicosanol; octatriacontyl pentafluoropropionate; 1-ioddodecane, octadecanoic acid, supraene and hentriacontane were significantly different between the collection periods (Table 1). In the second extraction solvent, 9-hexadecenoic acid pyrrolide; diclofop-methyl; 1-piperidin-1-yl-hexadecan-1-one; stigmasta-3,5-diene; ethyl glycolate; 1,2-dihydro-2,2,4-trimethylquinoline, palmitoleic acid, 9,12-(_Z_,_Z_)-octadecanenoic acid, methyl stearate and trimesitylborane were identified (Table 2). Principal component analysis (PCA), sparse partial least squares-discriminant analysis (sPLS-DA), and heat map and ANOVA analyses were used in these experiments. PCA visualizes both the covariance and correlation between the metabolites and the modeled class designation. Thereby the PCA-plot helps to identify statistically significant and potentially biochemically significant metabolites, based both on contributions to the model and their reliability. An extension of PCA, the sPLS-DA-plot, is applied to compare the outcome of multiple classification models compared to a common reference, e.g. control. The example used is a GC-coupled MS-based metabolomics study in extracted samples where two mating time lines are compared between extract solvents.