Development and Validation of Novel High‐Performance Liquid Chromatography and High‐Performance Thin‐Layer Chromatography Analytical Methods for the Determination of Signature Phytochemicals in Classical Centuries‐Old Natural Medicine, ChandraPrabha

ABSTRACT Ayurveda, often referred to as the “Science of Life,” offers a comprehensive approach to promote long, healthy lives, with its holistic principles contributing to the growing interest in Ayurvedic medicines. One such ancient Ayurvedic remedy is ChandraPrabha Vati (CPV), traditionally used to treat joint pain, urinary tract infections, bladder issues, and general weakness. However, to integrate these remedies into mainstream medicine, their standardisation is vital. Yet, a detailed standardization and scientific validation of this classical formulation remain scarce. The major objective of the current study is to establish robust and precise analytical techniques for the quantification of active phytochemicals in CPV using high‐performance liquid chromatography (HPLC) and high‐performance thin‐layer chromatography (HPTLC). The optimised methods of both analytical techniques were validated by following the International Council on Harmonization Q2 (R1) guidelines. The validation parameters included system suitability, limit of detection, limit of quantification, linearity, precision, recovery, and robustness. The results confirmed that both methods were linear (correlation coefficient >0.99) and precise for the quantification of the active ingredients. The study reported the marker‐based identification and quantification of significant phytochemicals present in CPV. Seven phytochemicals were quantified simultaneously using both HPLC and HPTLC: gallic acid, chebulagic acid, chebulinic acid, benzoic acid, guggulsterone E, guggulsterone Z, and piperine. In addition, compounds including, corilagin, ellagic acid, berberine, bisdemethoxycurcumin, demethoxycurcumin, and curcumin were quantified using HPLC. Among the identified compounds, benzoic acid was found to be the predominant component, followed by gallic acid. A comparative analysis of the phytoconstituents in CPV using both HPLC and HPTLC methods was also statistically conducted. This robust study methodology, coupled with the validation and quantification, offers valuable insights into the complex composition of the polyherbal formulation, CPV, helping to better understand its potential health benefits.