Development and Validation of an Optimized HPLC-DAD Method for Simultaneous Quantification of 14 Bioactive Compounds...

Lonicerae Flos, an important traditional Chinese medicine, possesses a complex chemical composition and variable bioactive compound profiles, thereby necessitating reliable analytical methods for its quality control. In this study, a novel high-performance liquid chromatography-diode array detection method was developed for the simultaneous quantification of 14 bioactive compounds in Lonicerae Flos. A two-stage optimization strategy was employed, combining gradient elution refinement with response surface methodology based on a Box-Behnken design to systematically optimize injection volume, column temperature, and flow rate. Under the optimal conditions, baseline separation of all analytes was achieved within 25 min. The validated method exhibited excellent linearity (R² > 0.9990), sensitivity (LOQ ≤ 0.40 μg/mL, LOD ≤ 0.12 μg/mL), precision (intra- and inter-day RSD ≤ 3.59%), and recovery (97.03-102.62%), with satisfactory stability over 24 h. When applied to Lonicerae Flos samples from three geographical regions, the method revealed significant variations in chlorogenic acid, neochlorogenic acid, and 3,4-dicaffeoylquinic acid, highlighting its utility for geographical origin tracing and regional quality assessment. Specifically, chlorogenic acid showed a gradual increasing trend from Xiushan and Suiyang to Longhui, whereas neochlorogenic acid and 3,4-dicaffeoylquinic acid reached their highest levels in Longhui samples. Compared with existing methods, the proposed approach demonstrates superior performance with wider analyte coverage, improved separation efficiency, and simplified sample pretreatment. Accordingly, this work provides a practical tool to address the key challenges in the quality control of this medicinal plant, particularly the complex and variable profiles of bioactive constituents, thus supporting the standardization and quality evaluation of Lonicerae Flos.