Sequential Extraction. The model organism C. elegans strain PD1074, a variant of the original N2 Bristol strain, was used throughout this study. Samples were sequentially extracted in triplicate, see Table (where (i) indicates non-polar and (ii) polar specific levels). All solvent mixtures were cooled to -20°C. The specified volume (Table ) for each first-round extraction solvent (Table ) was pipetted into each tube containing homogenized worm material and beads, vortex mixed for 30 s, and stored on ice until solvent addition to all samples was complete. For samples requiring 3 mL of extraction solvent, 1 mL of solvent was added to the original homogenized tube, vortex mixed, and transferred by pouring to a new 5 mL tube. This step was repeated with an additional 1 mL of extraction solvent to thoroughly rinse and transfer any remaining homogenate out of the All samples were extracted on a rocker in a cold room (4°C) for the specified time in Table . Samples in 2.0 mL tubes were centrifuged (Eppendorf, model 5417C) for 10 m at 10,600 RCF, while samples in 5.0 mL tubes were centrifuged (Eppendorf, model 5072) for 20 min at 3,000 RCF. Each supernatant was then transferred via Pasteur pipet to a new tube, leaving behind the sample pellet, homogenization beads, and a small amount of solvent. Supernatants and pellets were placed in a Labconco Centrivap until complete dryness (approximately 3 h and 30 min, respectively). Sample pellets were dried to prevent solvent carryover into the second extraction. The dried pellets were then extracted for polar metabolites as specified in Table . Dried extracts were stored at -80°C until they were ready to be analyzed by LC-MS and NMR spectroscopy (details included in supplemental). Ultra-High-Performance Liquid Chromatography (UHPLC-MS) Analysis. Dried samples were reconstituted in 100 µL of reconstitution solvent (Table ) and vortex mixed for 30 s. Non-polar and polar sample reconstitution solvents are specified as (i) and (ii), respectively for each experiment (Table ). Tubes containing the reconstituted solution were centrifuged at 3800 RCF for 10 min and transferred to LC vials for UHPLC-MS analysis. UHPLC-MS analyses were performed using a Vanquish UHPLC (Thermo Fisher Scientific, Inc., Waltham, MA) coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA). An injection volume of 2 µL was used for both RP and HILIC chromatography. Both separations were analyzed in both positive and negative ionization modes See supplemental methods for detailed LC-MS parameters and settings.