Development and Validation of a Sensitive and Accurate LC-MS/MS Method for the Quantification of Creatinine in Rat Urine

Creatinine which is produced during muscle energy production serves as an indirect marker of kidney function in clinical chemistry and as a parameter for urine matrix integrity in forensic toxicology.Urinary creatinine concentration is also used as a normalization factor for individual differences in drugs and metabolites in bioanalysis.Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) has shown considerable potential for the quantification of creatinine in urine samples, but sufficient validation has not been performed.Therefore, herein, we aimed to develop and validate a sensitive and accurate quantitative method for measuring creatinine levels in rat urine using LC-MS/MS.Water was used as a surrogate matrix for rat urine, and its suitability was fully demonstrated.The validation results demonstrated that the method was selective, sensitive, accurate, and precise, with acceptable linearity within a defined calibration range.The limit of detection and lower limit of quantification were 5.0 and 50 g/mL, respectively.In the urine samples of healthy rats (n = 6), the concentrations of creatinine, as determined via the developed method, ranged from 116.1 to 445.8 g/mL.This method is expected to be highly beneficial for quantitatively determining the levels of drugs and metabolites in rat urine.