Figure shows that the S/N for RP increases as extraction volume increases, while the S/N for HILIC decreases. This likely results from compounds that are present in both the non-polar and polar extractions. At lower extraction volumes, solutions can become saturated leading to an inefficient extraction with < 100% recovery and a percentage of more concentrated compounds remaining in the non-polar extraction pellet. When the polar extraction is conducted, the remaining amount of these saturated compounds can be extracted, if soluble, in the selected polar extraction solvent. As the saturation of the initial extraction increases, so does the number of features detected in both RP and HILIC, which can result in duplicate feature counts and increased HILIC S/N values. This is shown in Figure , where 0.5 mL of extraction solvent was used, and the highest S/N value for HILIC is observed. As the extraction volume increases in B/2 and B/3 we observe that the S/N of the HILIC extract decreases. For this reason, the RP and HILIC extractions in each main effects plots should be considered as pairs.