Development and Validation of RP-HPLC Method for Simultaneous Estimation of Luteolin, Ursolic Acid, Naringin, and Quercetin

Abstract

A simple, rapid, precise, and robust reverse phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous estimation of Luteolin, Ursolic acid, Naringin, and Quercetin in herbal formulation of Carissa Carandas L. Chromatographic separation was achieved using a Agilent HPLC-PDA and Hypersil ODS, C18 column (4.6 × 250 mm, 5 µm) with a mobile phase consisting of acetonitrile and water (90:10 v/v) at a flow rate of 1.0 mL/min. Detection was carried out at 210 nm using a UV detector. The developed method was validated according to ICH Q2(R1) guidelines for specificity, linearity, precision, intermediate precision, and robustness. The calibration curves exhibited good linearity over the concentration range of 25–150 ppm with correlation coefficient values greater than 0.99 for all analytes. Precision studies demonstrated %RSD values below 2%, indicating acceptable repeatability and reproducibility of the method. Robustness studies revealed that deliberate changes in wavelength and flow rate did not significantly affect chromatographic performance. The developed RP-HPLC method was found to be specific, accurate, precise, and robust, and can be successfully applied for routine quality control analysis of herbal formulations containing selected phytoconstituents.