Development and Validation of a Robust HPLC Method for Simultaneous Quantitative Analysis of Quercetin and β-sitosterol in Plant Extract

Quercetin and beta-sitosterol (β-sitosterol) are prominent phytoconstituents that are being widely used in food ingredients and medicinal applications due to their various pharmacological activities and therapeutic activities including anti-inflammatory, anti-cancer, and anti-diabetic properties. The main aim of this paper is the development and validation of a robust reverse phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of quercetin and β-sitosterol. Gradient elution was performed using a mixture of water, acetonitrile and acetic acid, with varying ratios of water:acetonitrile:acetic acid (50:50:2). The flow rate was set at 0.9 mL/min, and the detection wavelength was fixed at 370 nm. The developed method was validated according to the International Conference of Harmonization (ICH) Q2 (R1) guidelines. The method was linear in the concentration range of 1–5 µg/mL quercetin and β-sitosterol. The limit of detection (LOD) and limit of quantification (LOQ) were 0.118 and 0.358 µg/mL for β-sitosterol, respectively, while LOD and LOQ for quercetin were 0.127 µg/mL and 0.387 µg/mL respectively. The percentage recovery ranged from 95 to 105% with a relative standard deviation of less than 2%, indicating the accuracy and precision of the method for both compounds. Furthermore, the validated method was found specific to detect presence of both the drugs in Nyctanthes arbortristis leaf extract. The developed method is the first of its type method that is capable of simultaneous estimation of β-sitosterol and quercetin from Nyctanthes arbortristis leaves and demonstrated with excellent specificity, linearity, accuracy and precision. Consequently, it holds great potential for the detection of β-sitosterol and quercetin in food, biological samples and various marketed formulations.