Determination of uric acid in human serum by an enzymatic method using N-methyl-N-(4-aminophenyl)-3-methoxyaniline reagent

Abstract

In this work a new enzymatic method for the determination of uric acid in human serum has been developed. The method is based on the oxidative coupling reaction between the N-methyl-N-(4-aminophenyl)-3-methoxyaniline (NCP) reagent and the hydrogen – donor reagent, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), in the system involving three enzymes: uricase, peroxidase and ascorbate oxidase. Using this method uric acid could be determined in concentrations up to 1.428 mmol/L, with a relative standard deviation of up to 1.8 %. The effect of the medium pH and the NCP concentration on the linearity of the chromogen absorbance versus the uric acid concentration curve was investigated. The influence of the uricase activity on the maximum rate of uric acid oxidation was also examined. The use of the NCP reagent demonstrated a more precise and more sensitive determination of the uric acid compared to the determination with 4-aminoantipyrine (4-AA) as the coupling regent. The sensitivity of the method determined from the calibration curve was 0.71 absorbance units per mmol/L of uric acid; the limit of detection was LOD = 0.0035 mmol/L and the limit of quantification was LOQ = 0.015 mmol/L of uric acid.

Keywords

• uric acid
• human serum
• enzymatic determination
• NCP reagent