Development and Validation of an UPLC Method for Quality Control of Plantago major

Plantago major L. and Plantago australis Lam., Plantaginaceae, have been used in folk medicine mainly as anti-inflammatory and healing agents. The two species, known by the same popular names, are morphologically similar and frequently P. australis appears as adulterant or substitute of P. major. In this study, an efficient and reproducible extractive method was developed for quality control of P. major samples, capable of identifying differences in the chromatographic profile of both plants. A reflux extraction has been proposed and optimized by fractional factorial design and subsequent Box-Behnken design. The following conditions were defined to evaluate the verbascoside content: particle size 180 µm, without solvent renewal, mass solvent ratio in 1:60, percentage of solvent in 89%, and time in 60 min. A UPLC method was validated and allowed the determination of verbascoside content in P. major leaves reliably and in accordance with regulatory agency guidelines. Chromatographic profiles obtained by densitometry showed an average retention factor (R_f) of 0.77 for verbascoside. Comparison of the UV spectrum obtained by UPLC and densitometry showed that the peaks at retention time (R_t) 4.62 min and the bands at R_f 0.55, present in both species, belong to different compounds. Therefore, in accordance with the guidelines of RDC No. 166/2017, the results presented in this study proved that the proposed method was able to differentiate the material of interest from another similar plant species. This distinction makes it possible to use the method as a means of detecting adulteration or substitution by the industry in the absence of the desired raw material.