Simultaneous Analysis Method for Rutin, Diosmin, Hesperidin, and Quercetin in Solid Food Supplements by HPLC-PDA

Flavonoids are important component of dietary supplements, playing major roles as antioxidants, anti-aging agents, and numerous health benefits. The aim of this study was to develop a method for the simultaneous analysis of four flavonoids - rutin, diosmin, hesperidin, and quercetin in dietary supplements. A high-performance liquid chromatography (HPLC) method with reversed-phase separation and photodiode array detector was developed to simultaneously quantify rutin, diosmin, hesperidin, and quercetin in solid dietary supplements. The method used a Phenomenex Gemini C18 column (250 mm × 4.6 mm, 5 μm), controlled at 60°C. Separation was performed using a gradient mobile phase of acetonitrile and phosphoric acid (0.1%) at a 1.0 mL/min flow rate. Detection was executed at 284 nm for hesperidin and 254 nm for rutin, diosmin, and quercetin. Under the optimized chromatographic conditions, the method linearity was verified over the following concentration ranges: 6.2 – 794.0, 3.4 – 434.0, 3.3 – 420.0, and 3.2 – 414.0 μg/mL for rutin, diosmin, hesperidin, and quercetin, respectively. The method was validated for accuracy, linearity, and precision. The method exhibited high accuracy, with recoveries of 99.95±0.43, 100.08±0.42, 100.12±0.32, and 100.23±0.44% for rutin, diosmin, hesperidin, and quercetin, respectively. The relative standard deviation (RSD, %) of all four flavonoids did not exceed 1%. The method's robustness and simplicity make it well-suited for routine quality control of herbal products containing these flavonoids. The results showed that the method is a valuable tool for ensuring the quality consistency of dietary supplements containing rutin, diosmin, hesperidin, and quercetin, frequently used for human health benefits.