## 3 Results
### Multivariate Statistical Analysis  
The main purpose of this experimental trial was to establish the chemical fingerprints of industrial recycled RM processed using an ethanol-based extraction (i.e., EWS samples) and the further changes due to an optimisation treatment based on the use of ultrasound (i.e., OEWS samples). To this aim, a partial least square-discriminant analysis (PLS-DA) was carried out to allow for a graphical demonstration of the potential of DART-HRMS to capture the chemical signatures of EWS and of the optimised derivates (OEWS), as reported in Figure 2.  
As reported in Figure 2A, the spatial separation and clustering of the two groups of samples is defined by the PLS-DA. Precisely, the total variance of the model is explained by the three components C1 (55.3%), C2 (18.0%), C3 (17.2%) which are defined in the three-dimensional space. The differences in the metabolic fingerprints of the two rapeseed-derived matrices observed in the spectra (Figure 1) are confirmed by the graphical spatial discrimination. The outcomes of the PLS-DA were validated by LOOCV as shown in the Supplementary Materials (Figure S1). Specifically, the performance measures of the LOOCV, using five components and expressed as accuracy = 1.00, Q\({}^{2}\) = 0.91, and R\({}^{2}\) = 0.98, are also summarised in Figure S1. Figure 2B illustrates the most helpful fifteen ions (\(m/z\) values),  
Figure 1: Representative spectra of rapeseed meal (RM) ethanol-washed solutes, EWS (**A**) and their optimised derivatives, OEWS (**B**), acquired by direct analysis real time–high resolution mass spectrometry (DART-HRMS) in negative ion mode. The tentative annotation of the ions characterising the EWS are reported in green; those of OEWS are reported in red. together with their coefficient values, that aid the separation into the two groups. The box-whisker plots of the fifteen ions are reported in Figure 3 and their tentative annotations are listed in Table 1. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Observed \(m/z\)** & **Theoretical \(m/z\)** & **Error (ppm)** & **Predicted** & **Ion** & **Tentative** \\  & & & **Molecular** & **Type** & **Annotation** \\  & & & **Formula** & & \\ \hline  & & & EWS & & \\
117.0194 & 117.0193 & 0.9 & C\({}_{4}\)H\({}_{6}\)O\({}_{4}\) & [M\(-\)H]\({}^{-}\) & Succinic acid \\
133.0141 & 133.0142 & \(-\)0.8 & C\({}_{4}\)H\({}_{6}\)O\({}_{5}\) & [M\(-\)H]\({}^{-}\) & Malic acid \\
137.0269 & \(-\) & \(-\) & \(-\) & \(-\) & N/A \\
160.0404 & 160.0404 & 0.0 & C\({}_{9}\)H\({}_{7}\)NO\({}_{2}\) & [M\(-\)H]\({}^{-}\) & Indole-\(\beta\)-carboxylic acid \\
164.0355 & 164.0348 & 4.3 & C\({}_{8}\)H\({}_{9}\)NO\({}_{4}\) & [M\(-\)H\({}_{2}\)O\(-\)H]\({}^{-}\) & 4-Pyridoxic acid \\
185.0357 & 185.0348 & 4.9 & C\({}_{5}\)H\({}_{6}\)N\({}_{6}\) & [M\(+\)Cl]\({}^{-}\) & 2,6-Diaminopurine \\
255.2335 & 255.2330 & 2.0 & C\({}_{16}\)H\({}_{32}\)O\({}_{2}\) & [M\(-\)H]\({}^{-}\) & Palmitic acid \\
281.2486 & 281.2486 & 0.0 & C\({}_{18}\)H\({}_{34}\)O\({}_{2}\) & [M\(-\)H]\({}^{-}\) & Oleic acid \\ \hline  & & & OEWS & & \\
89.0242 & 89.0244 & \(-\)2.2 & C\({}_{3}\)H\({}_{6}\)O\({}_{3}\) & [M\(-\)H]\({}^{-}\) & Lactic acid \\
135.0305 & 135.0308 & \(-\)2.2 & C\({}_{2}\)H\({}_{6}\)S\({}_{2}\) & [M\(-\)H]\({}^{-}\) & Dimethyldisulphide \\
151.026 & 151.0261 & \(-\)0.7 & C\({}_{5}\)H\({}_{4}\)N\({}_{4}\)O\({}_{2}\) & [M\(-\)H]\({}^{-}\) & 6,8-Dihydroxypurine \\
187.0974 & 187.0976 & \(-\)1.1 & C\({}_{9}\)H\({}_{16}\)O\({}_{4}\) & [M\(-\)H]\({}^{-}\) & Azelaic acid \\
161.0444 & 161.0450 & \(-\)3.7 & C\({}_{6}\)H\({}_{12}\)O\({}_{6}\) & [M\(-\)H\({}_{2}\)O\(-\)H]\({}^{-}\) & Hexose \\
223.0612 & 223.0612 & 0.0 & C\({}_{11}\)H\({}_{12}\)O\({}_{5}\) & [M\(-\)H]\({}^{-}\) & Sinaptic acid \\
295.2279 & 295.2279 & 0.0 & C\({}_{18}\)H\({}_{32}\)O\({}_{3}\) & [M\(-\)H]\({}^{-}\) & Vernolic acid \\ \hline \hline \end{tabular} EWS, ethanol-wash solutes; OEWS, ultrasound-assisted optimised EWS; N/A, not assigned.  
\end{table}