RP-HPLC Method Development and Validation for the Marker Compound Embelin in Palashabijadi Churna

Abstract

Aim and Objectives: The present study aimed to prepare Palashabijadi Churna under laboratory conditions and to develop and validate a reverse-phase high-performance liquid chromatography (RP-HPLC) method for the quantitative estimation determination of embelin as , a marker compound. Methods: Palashabijadi Churna was prepared in accordance with the standards prescribed in the Ayurvedic Pharmacopoeia. RP-HPLC was then used to analyse the prepared formulation for the quantitative determination of embelin. Several preliminary chromatographic trials were performed to optimize the analytical conditions. The optimised mobile phase consisted of methanol and 0.1% orthophosphoric acid in a ratio of 90:10 (v/v), using a C-18 column. The analysis was carried out at 290 nm, with the column temperature maintained at 40°C. Before HPLC analysis, embelin was preliminarily identified by thin-layer chromatography (TLC) using a standard reference compound. Results: Embelin was detected in the laboratory-prepared formulation at an Rf of 0.32 by TLC using toluene: acetone: acetic acid (4.5:0.5:0.25, v/v/v) as the mobile phase and silica gel GF254 as the stationary phase. The λmax of embelin was found at 290 nm. In the RP-HPLC analysis, the standard embelin showed a retention time of 8.13 minutes, whereas the formulation sample exhibited a retention time of 8.07 minutes, thereby confirming its presence. Using the optimised mobile phase consisting of methanol and 0.1% orthophosphoric acid (90:10, v/v), the embelin content in the prepared formulation was quantified as 0.19% w/w. The developed RP-HPLC method was validated according to standard analytical validation parameters and demonstrated precision, accuracy, and reliability. Conclusion: The laboratory-prepared Palashabijadi Churna was systematically standardized through marker-based evaluation. The validated RP-HPLC method was found suitable for routine quantitative analysis of embelin, thereby supporting the quality control and standardization of the formulation.