Liquid Chromatography‐Mass Spectrometry Profiling and High‐Performance Liquid Chromatography Method Validation for Quantification of Acteoside, Emodin, and Chrysophanol in a Thai Traditional Anti‐Hemorrhoidal Formulation

ABSTRACT The phytochemical constituents of a Thai traditional anti‐hemorrhoidal formulation (TTAHF) remain insufficiently characterized, despite their importance for quality control. Therefore, this study aimed to investigate the liquid chromatography–mass spectrometry (LC‐MS) profiling and quantification of key compounds in TTAHF. LC‐MS analysis revealed that the most abundant compounds in the negative ionization mode were acteoside, emodin, quercitrin, kaempferol‐3‐O‐α‐L‐rhamnopyranoside, and chrysophanol. In contrast, the predominant compounds in the positive ionization mode were acteoside, chrysophanol‐8‐O‐glucoside, chrysophanol, acacetin, and stigmasterol. Three key compounds—acteoside, emodin, and chrysophanol—were selected as marker compounds for TTAHF. A high‐performance liquid chromatography (HPLC) method was validated for the quantification of these markers. The method demonstrated linearity, specificity, precision, and accuracy. Quantitative analysis of the TTAHF extract using the validated HPLC method showed that it contained 0.51% acteoside, 0.11% emodin, and 0.57% chrysophanol. In conclusion, this study provides LC‐MS profiling data and successfully establishes a validated HPLC method for the quantification of three marker compounds in TTAHF, which can be applied for the quality control of this traditional formulation.