Enzymic creatinine assay: a new colorimetric method based on hydrogen peroxide measurement

Area of Science:

• Clinical Chemistry
• Biochemistry
• Analytical Chemistry

Background:

• Accurate measurement of creatinine is essential for assessing kidney function.
• Existing methods may have limitations in speed, accuracy, or cost.

Purpose of the Study:

• To develop and validate a novel colorimetric method for quantifying creatinine in biological samples.
• To establish a rapid, accurate, and reliable assay for serum and urine creatinine.

Main Methods:

• Enzymatic hydrolysis of creatinine to creatine.
• Coupled enzymatic assay involving creatine amidinohydrolase and sarcosine oxidase to generate hydrogen peroxide.
• Colorimetric detection of hydrogen peroxide at 510 nm using horseradish peroxidase and a chromogen (3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone).

Main Results:

• The assay is complete within 30 minutes at room temperature.
• Linear standard curve for creatinine concentrations up to 2.21 mmol/L.
• Excellent analytical recovery (average 99.8%) in human serum and urine.
• High precision with coefficients of variation (CVs) ≤ 3.3% (within-run) and ≤ 4.3% (between-run) at 69 µmol/L creatinine.
• Strong correlation (r > 0.99) with established methods (enzymic UV and Jaffé).

Conclusions:

• The developed colorimetric method provides a reliable and accurate means for creatinine determination.
• This assay is suitable for routine clinical laboratory use due to its speed, precision, and agreement with existing standards.