Optimized and validated HPLC method for simultaneous quantification of rutin and verbascoside in multicomponent herbal mixture for wound healing

Rutin (a flavonoid glycoside) and verbascoside (a phenylethanoid glycoside) are bioactive phytochemical constituents widely recognized in traditional and modern phytotherapy for their antioxidant, anti-inflammatory and antibacterial activities. The presence of both rutin and verbascoside in a multicomponent herbal mixture (Verbena officinalis L., Hypericum perforatum L., Achillea millefolium L. and Plantago major L.) is expected to enhance its therapeutic efficacy in wound healing. In this study, an HPLC-DAD method was developed and optimized for the simultaneous determination of these two marker compounds within a single analytical run. The method was confirmed for selectivity, specificity, linearity, accuracy, precision, robustness, detection and quantification limits, as well as the stability of the standard mixture. Complete chromatographic resolution (Rs > 1.5) was achieved for both analytes. Excellent linearity (R2 ≥ 0.999) was achieved for both compounds. Accuracy was verified at 50 %, 100 %, and 150 % of the working concentration, while precision was demonstrated by relative standard deviation values below 2 %. Robustness testing showed consistent performance under minor variations in chromatographic conditions. Low detection and quantification limits were obtained for both analytes, and the standard mixture remained stable for up to 24 hours at room temperature. The validated method provides a rapid, reliable, and cost-effective tool for routine quality control and standardization of complex multicomponent herbal formulations.