Development and validation of a method for simultaneous determination of pseudouridine and creatinine in human urine: Evaluation's Index Pseudouridona/Creatinine in smokers and no-smokers

introduction: the excretion of pseudouridine is increased in inflammatory processes related to a muscle mass loss found in patients with pulmonary involvement. material and methods: a rapid and sensitive method for cuantification and simultaneous determination of pseudouridine, a breakdown product of trna, and creatinine in human urine via hplc was developed and validated. the mobile phase was 0.01 mol phosphate buffer (ph 6.1) containing 3 mmol octanesulphonic acid as ion pairing agent. sample preparation is based on dilution and filtration. a lichros-pher？ 100 rp-18 (5 μm) lichrocart？ 250-4 (merck) column with precolumn lichrospher？ 100 rp-18 (5 μm) lichrocart？ 50-4 (merck) were used, flow rate of 1ml/min. detection wavelength was set at 250 nm. results: the analysis time was 17 min per sample. the calibration range of pseudouridine (psu) and creatinine (crea) were 0.23-22.5 and 11.45-1100 nmol/ml. the linearity of the method was r2 = 0.997 and 0.998 and the lower limit of quantification (lloq) was 0.175 and 8.59 nmol/ml respectively. the average recovery (%) was 95.55 for pseudouridine and 97.82 for creatinine by addition and 93.16 and 89.79 % by dilution. the estimation of the coefficients of variation were < 8% for all levels. conclusions: a positive correlation was found between expected and observed values (pearson correlation coefficient = 0.99 for pseudouridine and 0.99 for creatinine). a correlation was found between recovery of pseudouridine and recovery of creatinine (pearson correlation coefficient = 0.86). this method was used to assess pseudouridine excretion in 30 healthy subjects (18 non-smokers and 12 smokers). there were no statistically differences between non-smokers and smokers.