Development and validation of a green AQbD based RPHPLC method for quantitative estimation of ascorbic acid in nutraceutical formulations

An analytical quality-by-design (AQbD)-based RP-HPLC method was developed to quantitatively determine ascorbic acid in nutraceutical formulations, guided by the principles outlined in ICH Q14 and validated using the applicable analytical procedure performance characteristics described in ICH Q2(R2). A Box–Behnken design (BBD) was used to evaluate how mobile-phase composition, flow rate, and pH affect key chromatographic characteristics, namely retention time, tailing factor, and theoretical plate count. Separation was performed on a ZORBAX Eclipse XDB-C18 column (150 × 4.6 mm, 5 μm) by using 0.05 M sodium dihydrogen phosphate buffer: methanol (80:20, v/v) at a flow rate of 1.0 mL/min, with UV detection set at 242 nm. Under the optimized conditions, ascorbic acid eluted as a well-resolved peak at approximately 1.77 min, within a total run time of 5 min. The method exhibited satisfactory linearity across 50–150 µg/mL (R² = 0.9976), with recovery close to 100% and precision and intermediate precision both below 2% RSD. The estimated limits of detection and quantification were 1.54 and 4.66 µg/mL, respectively. The procedure was applied successfully to marketed nutraceutical products, including tablets, multivitamin preparations, and oral drops. Its environmental impact was assessed using AGREE, GAPI, and NEMI, yielding an AGREE score of about 0.67, which points to a comparatively favourable environmental profile. Overall, the developed method demonstrated satisfactory analytical performance and practical suitability for determining ascorbic acid in the studied nutraceutical formulations.