A sequential extraction was used, where non-polar metabolites for RP analysis were extracted, followed by a polar metabolite extraction on the remaining compounds for HILIC analysis. Due to the broad XlogP range of each solvent, we anticipated a significant overlap between the two sequential extractions at low volumes. Since the nonpolar extraction was carried out first (to remove lipid-like molecules which can interfere with HILIC chromatography) and the HILIC extraction solvent was consistent for all nine experiments, we can observe the direct impact of the choice of non-polar extraction solvent on the results of the polar extraction. Figure shows the largest difference between RP and HILIC. Due to the difference in polarity between IPA and 80/20 MeOH/H2O, we can anticipate little overlap between the two solvents and observe a decrease in RP S/N and an increase of HILIC S/N in respect to the grand mean. When utilizing solvents of similar composition (Figure -A/2) MeOH and 80/20 MeOH/H2O, we can observe little to no change from the grand for RP and a decrease from the mean value in HILIC due to the overlap between solvents. Figure -A/3 demonstrates a middle-ground where we observe an increase from the mean for RP features extracted with a 1:1 1-Butanol:MeOH solution, which can solvate nonpolar and polar metabolite classes , but no change in the S/N in the HILIC extract in respect to the mean.