Supplementary Materials: Nonylphenol Toxicity Evaluation and Discovery of Biomarkers in Rat Urine by a Metabolomics Strategy through HPLC-QTOFMS

The HPLC system was equipped with a Waters XBridgeTM C18 column (2.1 × 150 mm, 5 μm), and the column temperature was set to 25 °C. The mobile phases for metabolic fingerprinting consisted of 0.1% formic acid in Milli-Q water and 5 mM ammonium acetate in Milli-Q water (solvent A, positive electrospray ionization (ESI+) and negative electrospray ionization (ESI–), respectively), acetonitrile (solvent B), and methanol (solvent C) in both (ESI+) and (ESI−) analyses. The following multi-step elution gradient was used: 0–2 min, 90% solvent A; 2–40 min, 90%–5% solvent A, which was kept for 10 min; 50–51 min, 5%–90% solvent A, which was kept for 10 min and then changed back to the initial mobile phase rate; 40–50 min, 30% solvent B; 0% solvent B in other periods. The flow rate of the mobile phases was 0.3 mL/min. The sample injection volume was 5 μL for all experiments. The ion source was a separated ESI ion source in TurboSprayTM. In ESI+ mode, the initial parameters for metabolomics were as follows: ion spray voltage, 5500 V; nebulizing gas pressure (GS1), 60 psi; drying gas pressure (GS2), 50 psi; ion source temperature, 500 °C; focusing potential, 265 V; curtain gas pressure, 25 psi; declustering potential, 80 V. In ESI-mode, the ion spray voltage was −4200 V; the declustering potential was −60 V; the focusing potential was −265 V; the other parameter settings were the same with ESI+. At the same time, the TOF-MS and information-dependent acquisition (IDA) methods were used to collect MS and MS/MS spectra. The methods involved a TOF-MS experiment with spectra ranging from m/z 50 to 1200 for metabolomic analysis. Dynamic background ions were subtracted to acquire MS spectra, which were recorded with automatic collision energy. In this way, lowand high-energy fragment ions were both present in a single spectrum.