Quantitative Profiling of Phenolic Constituents in Hypericum perforatum L. via HPLC-PDA and HPLC-ECD: A Chemometric Approach.

(1) Background: Medicinal plants are widely used in folk medicine. Hypericum perforatum L. (St. John's wort) is a medicinal plant that is used domestically and exported to other countries. This study addresses the need to develop methods for determining the composition and content of St. John's wort to determine its biological activity. (2) Methods: High-performance liquid chromatography (HPLC) equipped with an Electrochemical Detector (ECD) and a Photodiode Array Detector (PDA) was employed to identify and quantify major phenolic compounds-gallic acid, catechin, epicatechin, hyperoside, quercetin, and hyperforin-in extracted and lyophilized St. John's wort flower; stem; and leaf samples. Key analytes exhibited linear responses across both detection systems, within a quantification range of 0.5-10 µg/mL. (3) Results: The PDA method, validated according to ICH Q2(R1) guidelines, demonstrated specificity, linearity, precision, and accuracy, with limits of detection (LOD) ranging from 0.24 to 0.61 µg/mL and limits of quantification (LOQ) between 0.26 and 0.62 µg/mL. PDA effectively identified gallic acid, epicatechin, hyperoside, quercetin, and hyperforin, although catechin was not detected. ECD yielded comparable compound levels across the samples. (4) Conclusions: The novelty of this study lies in identifying the influence of climatic factors associated with the altitude at which St. John's wort is grown on the content and ratio of biologically active components. Overall, the chemometric approach demonstrates the utility of raw chromatographic data in distinguishing samples by plant part and geographic origin; even when traditional compound-based comparisons may be limited.