LC-MS/MS Validation of Ellagic Acid Rhamnopyranoside as a Species-Specific Chemical Marker for Authentication of Synadenium glaucescens in Herbal Medicines

ABSTRACT The authentication of medicinal plants used in herbal products remains a major challenge due to morphological similarities among species, processing-induced loss of diagnostic characteristics, and the absence of reliable species-specific chemical markers. Synadenium glaucescens , an important medicinal plant widely used in Tanzania, lacks an analytically validated marker for routine authentication and quality control. This study aimed to validate ellagic acid rhamnopyranoside (EAR) and evaluate its suitability as a candidate species-specific marker for the authentication of S. glaucescens using a targeted and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform, thereby providing an evidence-based approach for marker-driven verification of this medicinal species in finished herbal products Chromatographic and mass spectrometric conditions were systematically optimized using multiple reaction monitoring (MRM) under positive electrospray ionization (ESI). The developed method was validated for linearity, sensitivity, precision, accuracy, matrix effect, and chromatographic performance in accordance with international analytical validation guidelines and was subsequently applied to a commercial herbal formulation. EAR exhibited characteristic fragmentation via dual MRM transitions (m/z 477→331 and 331→299), which were tracked to provide high analytical specificity for compound confirmation. The optimized LC-MS/MS method demonstrated excellent linearity (R² = 0.9983), satisfactory sensitivity, acceptable precision and accuracy, negligible matrix interference, and stable chromatographic performance. EAR was successfully detected and quantified (444.41 µg mL⁻¹) in the commercial herbal formulation, confirming the presence of S. glaucescens in the finished product. This study establishes ellagic acid rhamnopyranoside as a validated species-specific chemical marker for Synadenium glaucescens , providing the first robust analytical framework for the authentication, quality control, and standardization of S. glaucescens -containing herbal medicines, while offering a practical approach to strengthen the regulatory evaluation of herbal products