Standardization of Romanian Galeopsis tetrahit Leaf Extract in Verbascoside Using a Validated UHPLC-PDA Method

Galeopsis tetrahit L. (Lamiaceae) is a traditional European medicinal species rich in phenolic compounds, among which verbascoside is a key bioactive marker with strong antioxidant potential. This study reports the standardization of a G. tetrahit leaf extract in verbascoside using a fully validated UHPLC-PDA method developed according to ICH Q2(R2) requirements. Leaves of wild-grown G. tetrahit collected from southwest Romania flora were extracted with 70% ethanol, yielding 17.28% dry extract. Chromatographic identification of verbascoside was confirmed by retention time, UV-PDA spectra, and QDa mass spectrometry (m/z 623.3 [M-H]^-). The method showed excellent performance, including high specificity, linearity over 1.875-60 μg/mL (r = 0.999955), low LOD and LOQ (0.2649 and 0.8028 μg/mL, respectively), and robust precision and accuracy. Dry extract contained 345.8 ± 28.3 mg verbascoside per g (34.6%, w/w), corresponding to approximately 59.8 mg/g in dried leaves. Antioxidant assays (DPPH, ABTS, FRAP), TPC and TFC confirmed notable radical scavenging and reducing activity, with pure verbascoside showing markedly stronger effects, supporting its major contribution to the extract's antioxidant potential. These results demonstrate a reliable analytical approach and establish a verbascoside-based standardization framework for G. tetrahit extracts of documented Romanian origin.