## 2 Materials and Methods
The desorption solution was then diluted with 240 \(\mu\)L of water to produce a final extract composed of 1:1 organic/aqueous content (4:3:7 methanol:acetonitrile:water). A total of 10 \(\mu\)L of each sample extract was combined to form a pooled quality control (QC) sample that was injected in the analytical run every 10 sample injections. Chromotagraphic separation was conducted on an Accuocre C30 HPLC column (100 mm \(\times\) 2.1 mm, 2.6 \(\mu\)m) using the developed method. Gradient elution in positive mode was accomplished over a 30 min period using mobile phases composed of 99.9 water/0.1 formic acid (v/v) and 99.9 methanol/0.1 formic acid (v/v). Gradient elution in negative mode was accomplished over a 20 min period using mobile phases composed of 99.9 water/0.1 formic acid (v/v) and methanol. During analysis, 5 \(\mu\)L of each sample extract was injected, with the autosampler at 5 \({}^{\circ}\)C and the column temperature at 60 \({}^{\circ}\)C. Please see Tables 5 and 6 for additional details on the chromatographic gradient elution. The parameters for mass spectrometry were consistent with those outlined in the assay development. The lipids outlined in Table 3 were prepared in 4:3:7 methanol/acetonitrile/water and used as quality control standards throughout the LC-MS analysis. The LC-MS data files acquired during instrumental analysis were pre-processed on MetaboAnalyst 5.0. The raw LC-MS data files were first converted to mzML files using \begin{table}
\begin{tabular}{c c} \hline \hline
**Time (min)** & **\% Mobile Phase B** \\  & **(Methanol + 0.1\% Formic Acid)** \\ \hline
2 & 0 \\ \hline
12 & 100 (curve 3) \\ \hline
15 & 100 \\ \hline
18 & 0 \\ \hline
20 & 0 \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Liquid chromatographic gradient used for separation in negative mode. \begin{table}
\begin{tabular}{c c} \hline \hline
**Time (min)** & **\% Mobile Phase B** \\  & **(Methanol + 0.1\% Formic Acid)** \\ \hline
0 & 5 \\ \hline
1 & 5 \\ \hline
20 & 100 (curve 3) \\ \hline
22.5 & 100 \\ \hline
25 & 5 \\ \hline
30 & 5 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Liquid chromatographic gradient used for separation in positive mode. MSConvert [36] and then pre-processed using the MetaboAnalyst LC-MS Spectra Processing module [37]. The module performed automated parameter optimization based on pooled quality control (QC) samples run throughout the sequence and supported spectra data processing for peak picking, alignment, and gap filling [37]. The peak lists generated using MetaboAnalyst were initially filtered by removing features with a pooled QC relative standard deviation (RSD) greater than 30%. Further filtering was performed by removing features that had a pooled QC to solvent/device blank ratio of less than 5. The data was uploaded back onto MetaboAnalyst; default settings were used for missing values, with no supplementary filtering applied. A total of 1786 features were detected in positive mode and 1059 features in negative mode. Prior to univariate or multivariate chemometric analyses, a log transformation was performed on the negative mode data, and a cube root transformation in addition to pareto scaling was performed on the positive mode data. Statistical analysis was performed using MetaboAnalyst 5.0. Since the distribution of the data cannot be assumed, a Wilcoxon Rank test or a Kruskal-Wallis test, both with a false discovery rate (FDR) of 0.05, were applied for univariate analysis. Multivariate analysis included Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA), and Orthogonal Projection to Latent Structures-Discriminant Analysis (OPLS-DA). Model validation for O-PLS-DA and PLS-DA included a combination of permutation (\(p<0.05\) at 2000) and 10-fold cross-validation (10-fold CV). Only features with a variable of importance in projection (VIP) score of \(>\)1 were investigated further.