Rapid RP-HPLC method optimization for vitamin D3 quantification in soft-gelatin capsules via Response Surface Methodology (RSM)

Reliable quantification of fat-soluble vitamins such as vitamin D₃ (VD₃) in soft-gelatin dosage forms is challenging because of co-formulated antioxidants and variable excipients. A rapid and robust RP-HPLC method was optimized and validated for VD 3 quantification in soft-gelatin capsules containing Vitamin E as an antioxidant using response-surface methodology (RSM). A Box–Behnken design (BBD) within a Design of Experiments (DoE) framework was used to optimize column temperature, flow rate, and mobile phase composition. The final 5 min method employed an Acclaim RSLC 120 C18 column with ACN:MeOH:Milli-Q water (87.5:9:3.5) as the mobile phase, a flow rate 0.8 mL min −1 , and column temperature of 20.8 °C. Sample clean-up evaluation identified the SOLA HRP SPE cartridge as providing the highest recovery (98.83 ± 0.65%). Validation demonstrated specificity, linearity (0.02–6.00 mg L −1 , R 2 = 0.999), accuracy (recoveries 98.2–99.5%), precision (%RSD 0.66%), and sensitivity (LOD 2.5 μg L −1 , LOQ 6.7 μg L −1 ). This study highlights the utility of RSM-BBD for efficient HPLC method optimization, providing a generalizable strategy for other fat-soluble vitamins and complex formulations. • The study focuses on soft-gelatin capsules, an analytically challenging and underexplored dosage form. • A statistically optimized RP-HPLC method for vitamin D 3 quantification was developed. • Response Surface Methodology (RSM) was applied to systematically optimize key chromatographic parameters. • The method demonstrates excellent robustness, precision, and reproducibility across tested conditions. • The statistical design enabled efficient evaluation of factor interactions and analytical reliability. • This work provides a data-driven framework for method optimization in pharmaceutical analysis and quality control.