Background: Measurement of urinary creatinine has a diagnostic value in clinical practice, and is used for correction of urine sample dilution in metabolomics studies. Rapid and reliable methods for determination of urinary creatinine using routine laboratory instruments are greatly needed.

Results: The development of a convenient homogeneous enzyme immunoassay (EIA) for urinary creatinine has been described here. The working principle is: free creatinine competes for specific antibodies binding to creatinine-glucose-6-phosphate dehydrogenase (G6PDH) conjugate to derepress the enzyme activity, which is measured by increasing absorbance at 340 nm by reduced NAD (NADH) and is proportional to the concentration of free creatinine in a urine sample. The method showed an assay linear range of 1-5000 mg/L (R² = 0.990), and a limit of detection (95 % confidence) of 24.73 mg/L for urinary creatinine. The average recovery for spiked urine samples was 101 %. Both the intra-assay and inter-assay variations were less than 3 %. The proposed method was specific with no significant interference when analyzing urine samples containing high levels of the common interfering substances. Excellent concordance was observed between the current method and a conventional enzymatic method. The wide measurement range of the method allows the direct determination of creatinine in urine samples without time-consuming pretreatments or complicated procedures.

Significance: The method has great potential for routine use in clinical and research laboratories.

Keywords: Creatinine; Enzyme immunoassay; Homogeneous; Urine.

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