Formononetin and Naringenin Concurrent Measurement in Plasma Using RPHPLC Method Development and Validation

The present study aims to develop and validate a robust RP-HPLC method for the simultaneous estimation of naringenin and formononetin in human plasma. Chromatographic separation was achieved using a Phenomenex C18 column (250 mm × 4.6 mm, 5 μm) with an isocratic mobile phase of methanol:water (50:50, v/v) at a 1 mL/min flow rate. Levocetirizine was selected as the internal standard based on retention time compatibility. A protein precipitation method was optimized for sample extraction, and the method was validated for selectivity, precision, accuracy, and stability following regulatory guidelines. The method demonstrated excellent linearity (0.15–6 µg/mL) with a correlation coefficient (R²) > 0.999. The LLOQ was established at 0.15 µg/mL. Recovery studies confirmed efficient extraction with recoveries of 94.2% for naringenin and 91.5% for formononetin. Stability assessments, including bench-top and freeze-thaw studies, indicated that both compounds remained stable under different storage conditions. Precision and accuracy values were well within acceptance limits, ensuring the method's reliability. The developed RP-HPLC method provides a sensitive, accurate, and reproducible approach for the simultaneous quantification of Naringenin and Formononetin in plasma. This validated method is suitable for pharmacokinetic and bioavailability studies.