A High-Performance Liquid Chromatography Method for Simultaneous Analysis of Naringin, Ascorbic Acid, and Caffeine: Design, Development, and Validation.

The simultaneous quantification of ascorbic acid, caffeine, and naringin is of significant interest due to their potential therapeutic applications, particularly in the prevention and management of colorectal cancer. While numerous HPLC methods exist for the individual determination of each compound, our method stands out for its reliability in addressing all three compounds in a single analytical run. This study aimed to develop and validate a robust reverse-phase HPLC method for the simultaneous analysis of these compounds in accordance with ICH Q2(R1) and USP guidelines. A robust reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous determination of ascorbic acid, caffeine, and naringin in the concentration range of 7.813-125 μg/mL. Chromatographic separation was achieved on a Symmetry Shield RP-18 column (250 × 4.6 mm, 5 μm) using an isocratic mobile phase of 75% phosphate buffer (pH 3.5) and 25% acetonitrile at a flow rate of 1 mL/min, with detection at 220 nm. Method validation confirmed specificity, selectivity, and excellent linearity ( ≥ 0.9981). Precision testing showed RSD% values well below 2% for both intraday and interday analyses. Accuracy studies demonstrated mean recoveries within the range of 98%-102% (ascorbic acid: 100.05%, caffeine: 100.33%, and naringin: 100.47%). Robustness testing indicated that small deliberate changes in the flow rate and pH had no significant effect on retention times (RSD% ≤ 0.71%). System suitability parameters, including tailing factor (≤ 1.38), theoretical plates (> 2000), and resolution (> 3.2), met the acceptance criteria. The developed method is reliable, reproducible, and suitable for routine quality control applications that require the synchronized quantification of these three bioactive compounds.