2.4 Sample preparation for untargeted metabolomics
Ice-cold acetonitrile containing chlorpropamide (4 µM), atenolol-d7 (1.8 µM), flurazepam (0.15 µM) and DL-2-aminoheptanedioic acid (100 µM) as internal standards, was added to plasma and urine samples for protein precipitation (3:1 ratio). Kidney samples (50 mg) were homogenized in 150 µL of ACN containing internal standards. Following the addition of ACN, all samples were vortexed, incubated at -20°C for 20 minutes, and centrifuged at 14000 g for 10 minutes. Supernatant from the protein precipitation was diluted 1 in 5 with water. Diluted samples were transferred into glass vials for UPLC-MS analysis. A pooled sample was generated to serve as quality control throughout the metabolomics run.