PCA was used as an unsupervised approach to investigate the reproducibility of extraction replicates of the nine experimental runs. Figure shows the PCA scores plot for reversed-phase positive mode (RP+) extracts. Scores plots for the other LC-MS conditions can be found in Supplemental Figure -S4. PC1 and PC2 accounted for 25.4% and 12.6% of the variation within the dataset, respectively. Additional principal components did not further separate any of the nine groups and therefore were omitted. 95% confidence intervals (CI) are represented by the matching color-filled ellipses. All runs showed tight clustering of the three individual replicates, indicating low variation arising from sample growth, sample preparation, and instrument drift (Figure ). The clear separation between all nine runs suggests that the factors and levels chosen were appropriate and impacted the overall extraction performance. Samples were also clearly separated along PC1 by different extraction solvents with little overlap. No other factors clearly impacted the separation along PC1 or PC2. Run four showed the highest level of variation among replicates. However, this datapoint was not determined to be an outlier due to falling within the 95% CI. All 27 samples were included in the downstream analysis.