2.2. NMR sample preparation

Thirty-nine samples of urine from sixteen piglets before and after administration of boluses contaminated with a mixture of 5 mycotoxins were obtained as described above and analyzed by ¹H NMR spectroscopy. Frozen samples were thawed at room temperature and shaken before use. Aliquots of each urine sample (630 μL) were added to 70 μL of potassium phosphate buffer (1.5 M K₂HPO₄ in D₂O, pH 7.4), to minimize variations in metabolite NMR chemical shifts arising from differences in urinary pH, also containing 0.1% sodium 3-(trimethylsilyl)-[2,2,3,3-d₄]propionate (TSP) and 2 mM sodium azide. Samples were centrifuged at 14,000g for 5 min at 4 °C to remove any solid debris. 600 μL of the supernatant were placed in a 5 mm outer diameter NMR tube.