Comparison of Jaffe, enzymatic, and mass spectrometric methods for creatinine measurement in protein-positive and hemoglobin-positive urine samples

Background: Accurate measurement of urine creatinine is essential for evaluating renal function. Although the Jaffe and enzymatic methods are routinely applied in clinical laboratories, various urinary compounds can interfere with these assays. Methods: This study investigated the effects of hemoglobin and protein positivity on creatinine measurement and partially validated a mass spectrometric (LC–MS/MS) procedure. Urine samples from 136 participants were categorized based on dipstick results into three groups: negative (n = 55), hemoglobin-positive (n = 51), and protein-positive (n = 30). Creatinine concentrations were measured using Jaffe and enzymatic methods on an Architect c16000 analyzer and by LC–MS/MS on an API 3200 system. Results: The LC–MS/MS method demonstrated linearity between 3 and 350 mg/dL, intra- and inter-assay coefficients of variation of 3.2–3.8% and 1.9–2.9%, and bias values ranging from 1.1% to 18.2%. Correlations among LC–MS/MS, enzymatic, and Jaffe methods ranged from 0.981 to 0.998. Bland–Altman analysis showed mean absolute biases of 0.06–17.33 mg/dL (1.48–19.42%) in controls, 1.44– 4.78 mg/dL (0.79–9.59%) in hemoglobin-positive samples, and 2.18–9.87 mg/dL (0.52–14.39%) in protein-positive samples. Regression analysis showed slopes closer to unity in hemoglobin- and protein-positive samples compared to controls, likely reflecting higher creatinine concentrations in these groups. Despite this apparent improvement, bias values increased, particularly in protein-positive samples. Conclusion: Both routine methods showed acceptable agreement with LC–MS/MS, while the enzymatic method demonstrated slightly closer agreement under these conditions. LC–MS/MS maintained highest analytical specificity. These findings highlight the importance of method selection for reliable interpretation of renal function, especially in patients presenting with hematuria or proteinuria.