RESUMO
An analytical method was developed and validated for the quantitative determination of uric acid in cereals and pulses based on salting-out assisted extraction and subsequent analysis by Rapid Resolution Liquid Chromatography (RRLC). Uric acid is a degradation product of purines, which is an indicator of insect infestation and the state of stored grains and pulses. This study aims to compare and validate a high-performance liquid chromatography method with Diode-array detection (HPLC-DAD) and fluorescence detection (HPLC-FLD) for the estimation of uric acid. Protein precipitation with ammonium sulfate and acetonitrile was used for sample cleanup and pre-treatment. The addition of inorganic ions results in preferential solvation and precipitates proteins. The separation was performed on a Zorbax SB C18 column (150 × 4.6 mm, 5 µm) with an isocratic elution using water-acetonitrile containing 10 mM sodium dihydrogen phosphate (95:5, v/v), at a flow rate of 0.5 mL/min. The relative coefficient (r2) for the calibration curve was more than 0.995 over the concentration range of 25-200 mg/kg. This method's precision at concentrations of 25-150 mg/kg was within 7.25%, and the accuracy was 85.1%-92.7%. The method was validated in terms of the LOD, LOQ, repeatability, reproducibility, linearity, uncertainty, specificity & system suitability. The limit of detection and limit of quantification was 16.60 mg/kg and 50.34 mg/kg, respectively.
