5.4. LC-MS/MS Measurements for Indoxyl Sulfate and Creatinine

This measurement reference to the previous our reported methods [45]. For sample preparation, 150 μL of 0.1% formate methanol (containing 2.0 µg/mL creatinine-d₃ and 1.25 µg/mL indoxyl sulfate-d₄) were added to 50 μL of each plasma and vortexed for 1 s. The samples were then sonicated for 5 min and centrifuged at 16,400× g for 20 min at 4 °C. The supernatant was filtered through membranes (pore size: 0.22 μm; Merck Millipore, Billerica, MA, USA). Quantitative analysis of indoxyl sulfate was performed using LC-MS/MS using a Prominence LC system (Shimadzu, Kyoto, Japan) coupled to a TSQ Quantiva mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), and operated in negative mode. Each sample (5 µL) was injected onto a 150 × 2.0 mm YMC-Pack Pro C18, 3-µm column (YMC, Kyoto, Japan) at a flow rate of 0.3 mL/min, as we previously described [16]. For gradient elution, mobile phase A was 10 mM ammonium acetate, and mobile phase B was acetonitrile. Linear and stepwise gradients were programmed as follows: 0–1 min: 0–10% solvent B; 1–2 min: 10–40% solvent B; 2–3 min: 40–80% solvent B; 3–5 min: 80–100% solvent B; 5–7 min: 100% solvent B; 7–10 min: 0% solvent B. Quantification analyses by MS/MS were performed by a selected reaction monitoring mode (SRM), in which the transitions of the precursor ion to the product ion and collision energy (eV) were monitored: m/z 212→80, 21 eV for indoxyl sulfate; m/z 216→80, 30 eV for indoxyl sulfate-d₄. Quantitative analysis of creatinine was performed using LC-MS/MS, and operated in the positive mode. Each sample (5 µL) was injected onto a 150 × 2.0 mm YMC-Pack Pro C18, 3-µm column (YMC, Kyoto, Japan) at a flow rate of 0.2 mL/min. For gradient elution, mobile phase A was 0.1% formate in pure water, and mobile phase B was acetonitrile. Linear and stepwise gradients were programmed as follows: 0–1 min: 2–5% solvent B; 1–3 min: 5 –20% solvent B; 3–5 min: 20–50% solvent B; 5.1–7 min: 100% solvent B; 7–10 min: 2% solvent B. Quantification analyses by MS/MS were performed by a SRM mode, in which the transitions of the precursor ion to the product ion and collision energy (eV) were monitored: m/z 114→86, 11 eV for creatinine; m/z 117→89, 12 eV for creatinine-d₃. Spray voltage was 3000 V, vaporizer temperature was 350 °C, and ion transfer tube temperature 350 °C.