Analytical validation of a UV–Vis spectrophotometric method for precise and sensitive biochemical quantification

A UV–Visible spectrophotometric method was rigorously validated for precise and sensitive biochemical quantification, yielding excellent linearity, low detection limits, and high accuracy and precision across the full analytical range. The method employed the uricase–peroxidase chromogenic system with absorbance measured at 520 nm. Calibration was linear within 0.5–15.0 mg/dL (R² = 0.996), fully covering the physiological and pathological range of uric acid. The limit of detection (0.15 mg/dL) and limit of quantification (0.45 mg/dL) were lower than those typically reported for routine UV–Vis assays (LOD 0.3–0.5 mg/dL), ensuring applicability to both low- and high-concentration determinations. Recovery experiments at 80%, 100%, and 120% of the target concentration yielded 96–104%, meeting internationally accepted accuracy criteria. Precision was robust, with intra-day RSD values below 5% and inter-day values below 6%, surpassing the ≤10% variability commonly observed in conventional spectrophotometric assays. Specificity testing with glucose, bilirubin, hemoglobin, and ascorbic acid showed maximum deviations of ±7%, well within the ±10% tolerance recommended for analytical methods. Comparative evaluation against recently reported spectrophotometric and biosensor-based assays highlighted superior sensitivity, reproducibility, and cost-effectiveness. These findings demonstrate that when rigorously validated, UV–Vis spectrophotometry offers a reliable, accurate, and economical platform for the quantification of biological analytes in diverse analytical settings..