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Our aim was to validate a mass spectrometric creatinine method and compare this method with Jaffe and enzymatic serum creatinine methods. 90 samples were included. The levels were classified into three groups according to serum creatinine results as Group 1: Lower (n=30) (0.16–0.59 mg/dL), Group 2: Normal (n=30) (0.62–1.18 mg/dL) and Group 3: Higher (n=30) (1.33–3.88 mg/dL). Jaffe and enzymatic creatinine measurements were performed on the Beckman Coulter AU5800 autoanalyzer. Serum creatinine was linear from 0.039 up to 10 mg/dL, CV and bias values were ranged between 1.9–3.8% and 2–15%. Correlation coefficients were 0.990 (95% confidence interval 0.984–0.993), 0.992 (95% confidence interval 0.988–0.995) and 0.994 (95% confidence interval 0.991–0.996) for LC-MS/MS-Enzymatic, LC-MS/MS-Jaffe and Enzymatic-Jaffe, respectively. Although, Jaffe method for serum creatinine measurement is still much more practical and cheap, so in use for routine practice, tandem mass spectrometric detection of serum creatinine can be used as an accurate and specific method for verification of discordant clinical results, existence of possible interferences and serum levels under 0.5 mg/dL creatinine results such as pediatric or pregnant populations. [ABSTRACT FROM AUTHOR]