Determination of urinary creatinine

A 10 μL aliquot of each urine sample was first spiked with 10 μL of internal standard (100 ppm D₃-creatinine) and then diluted 1,000-fold with water before injecting 1 μL onto an ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 μm particle size). UPLC was performed at a flow rate of 0.4 mL/min using the following conditions: 100% solvent A (5 mM ammonium acetate) for 1 min, a liner gradient to 100% solvent B (100% acetonitrile) for 2 min, 100% B for 1 min, followed by re-equilibrium for 1 min with 100% solvent A. The mass spectrometer was operated in positive MS scan mode with cone voltage at 20 V, other MS operation condition were the same as described above for EXE and its metabolites. Standards at concentrations ranging from 0.002 to 2 ppm were used to establish standard curves for quantification of urinary creatinine levels.