4.4.1. Sample Preparation

The urine samples, previously stored at –80◦ C, were thawed on ice and centrifuged at 4000 rpm for 10 min at 4 ◦C; then, 500 μL of supernatant were collected. Then, 50 μL of phosphate buffer solution [41] (1.5 M K2HPO4/NaH2PO4, 30 mM NaN3, and 5.5 mM TSP, pH 7.4 in D2O) were added, and 50 μL of the final solution were transferred to a 1.7 mm thin-walled glass NMR tube for subsequent NMR analysis.
