Based on the main effects plots for the number of features and TIC, the optimal sequential extraction method (based on the factors/level combinations tested) for mass spectrometry-based untargeted metabolomics is shown in the last row of Table . No significant conclusions could be reached for factors with very low percent contribution and high variability, such as time and reconstitution solvent. As such, we relied on our current findings in conjunction with prior research findings and throughput requirements. For reconstitution solvent, we referred to separation science theory to determine the optimum conditions. Typically, a mixture of H2O and an organic modifier such as MeOH is used for RP chromatography. However, previous work by Lindahl et al. has shown pure H2O to be the ideal choice for untargeted metabolomics of human serum. For HILIC chromatography, Manier et al. showed increased performance with increasing MeOH for human plasma. Considering these previous studies and the results observed in our current study, a reconstitution solvent of 60/40 H2O/ACN and 80/20 MeOH/H2O was selected for RP and HILIC, respectively. For time, due to the high variability shown at 30 min (C/1) and 2 h (C/2), no clear conclusions could be made. However, the 12 h (C/3) extraction showed an increase in S/N and a decrease in variability of the individual measurements. This is likely due to an equilibrium reached during this longer extraction time. Based on these results, a 12 h extraction was deemed optimal. We recognize for larger study sizes requiring a high throughput approach a 12 h extraction is not ideal. The benefit of the Taguchi method is that factors are explicitly chosen to an investigator's needs and can provide preliminary results used to narrow down follow- Detailed information on these values can be found in Table .