Isolation-Driven UHPLC-DAD Method Validation and Multi-Regional Chemometric Profiling of Polyphenol Diversity in Rheum webbianum

In this study, six important polyphenols were isolated for the first time from the plant Rheum webbianum. Their structures were confirmed using NMR and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis. The study aimed to examine how the levels of these compounds vary in the roots of plants collected from different places. Since their concentrations can change with altitude and local environmental conditions, a clear and reliable analytical method was required. A simple RP-C18 ultra-performance liquid chromatography coupled with a diode array detector (UPLC-DAD) method was developed using an acetonitrile–water mobile phase containing 0.1% formic acid, with detection at 254 nm. The method was validated according to ICH guidelines. This work is among the first to compare these six compounds across multiple locations using both quantitative analysis and chemometric tools. The results showed noticeable variation in some marker compounds between regions, while a few remained relatively stable. This is the first report documenting the chemical diversity of R. webbianum across multiple regions of Ladakh. The developed method demonstrated good linearity, precision, and recovery. Overall, this study highlights the chemical diversity of R. webbianum across different locations and provides a reliable approach for its quality control and future research.