Validation and comparison of analytical methods for the determination of uric acid in pulses and cereals by salting out assisted extraction by Rapid resolution liquid chromatography

Abstract

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.