Liver Metabolomics Sample Preparation

Liver extracts were prepared based on previous methods [29 (link)]. Liver samples were pre-weighed (120–150 mg) and placed into MagNA Lyser tubes containing ~50 beads. Cold homogenization solution, 5× (5 μL/mg of tissue) (80:20 methanol: H₂O), was added to each tube, while keeping the samples frozen. Method blanks were prepared by adding 500 μL of homogenization solution to four empty MagNA Lyser tubes with beads and were processed identically to study samples. Samples were homogenized on an Omni Bead Ruptor Elite at 5 m/s for 30 s. Protein and tissue debris were pelleted by centrifuging samples at 4 °C and 16,000× g for 10 min. A volume of 200 μL of supernatant was transferred to new pre-labeled 2.0 mL low-bind Eppendorf tubes and dried by SpeedVac overnight. All samples were reconstituted by adding 500 μL of Tissue Reconstitution Solution (95:5 H₂O:methanol with 500 ng/mL Tryptophan-d5) and vortexing at 5000 rpm for 10 min on a multi-tube vortexer, followed by centrifugation at 4 °C and 16,000× g for 10 min. Supernatants were transferred to autosampler vials, and 5 μL of each study sample was combined to make a total QCSP. An injection volume of 5 μL was used for untargeted LC-MS analysis.