6545xt q tof

We mixed urine samples or calibration standards with creatinine‐D3 as an internal standard (IS) in a V‐bottom, polypropylene 96‐well plate, and then extracted by mixing with an extraction solution (75% acetonitrile/25% methanol at 1:3 ratio). The plate was covered with a lid and centrifuged at 5000g for 15 min at 4°C. The supernatant was transferred to a V‐bottom polypropylene plate, sealed, and then subjected to LC–MS analysis. Samples were injected via refrigerated autosampler into mobile phase, chromatographically separated by an Agilent 1290 Infinity II UPLC, and detected using an Agilent 6545XT Q‐TOF equipped with a dual jet stream ESI source operating under EDR (1700 m/z) in positive ionization mode. Table S2 provides chromatography and instrument conditions. MS1 spectra were collected in centroid mode, and peak assignments in samples were made based on comparisons of retention times and accurate masses from authentic standards using MassHunter Quantitative Analysis v.10.0 software from Agilent Technologies. We quantified creatinine concentration in urine samples from calibration curves constructed with an authentic creatinine standard using isotope‐dilution mass spectrometry with creatinine‐D3 as an IS.