To confirm these combined factors/levels outperform all other investigated runs, a triplicate extraction was carried out using the defined optimal levels. Samples were analyzed and processed in the same manner as all previous experiments allowing for comparison across different runs. Figure shows overlaid scatter and line plots for RP (+) and HILIC (+) of all nine runs (1-9) and the optimized experimental parameters. The inverse relationship between RP and HILIC is again observed due to the paired nature of the sequential extraction. The average number of total compounds for RP and HILIC combined was 3792 +/-1025. For both RP and HILIC, the optimized experiment resulted in the highest number of features for RP and the lowest number of features for HILIC. S/N values for the optimized experiment were also calculated and showed the same trend. These results indicate that by combining the optimum levels for each factor, we get a noticeable increase in the efficiency of our sequential extraction method with high overall coverage and a potential decreased chemical overlap between extractions. Overlaid scatter and line plots for negative mode and TIC are shown in Figures .