Development and Validation of an HPLC Method for Simultaneous Quantification of Diosgenin and Piperine in Plasma using RP-HPLC

Abstract

Introduction: The present study aims to develop and validate a robust RP-HPLC method for the simultaneous estimation of Diosgenin and Piperine in human plasma. Materials and Methods: Chromatographic separation was achieved using a Phenomenex C18 column (250 mm × 4.6 mm, 5 μM) with an isocratic mobile phase of methanol: water (70:30, v/v) at a 1 mL/min flow rate. Nintedanib 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. Results: 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 94.3% for Diosgenin and 91.5% for Piperine. 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. Conclusion: The developed RP-HPLC method provides a sensitive, accurate, and reproducible approach for the simultaneous quantification of Diosgenin and Piperine in plasma. This validated method is suitable for pharmacokinetic and bioavailability studies.