A gradient high-performance liquid chromatography (HPLC) method with diode array detection (DAD) was developed and fully validated in accordance with ICH guidelines, for the determination of 20-hydroxyecdysone. This bioactive compound is a highly polar phytoecdysteroid with potential health activity, and its reliable quantification in complex formulations represents a significant analytical challenge, particularly in the presence of lipid-based excipients commonly used in nanomaterial research. The developed approach can be considered universal for compounds of similar polarity, including hydrophobic derivatives and structurally related analogues, as well as hydrophilic substances.
Chromatographic separation was achieved on a reversed-phase C-18 column using a binary mobile phase system composed of phase A: 0.1% (v/v) phosphoric acid in water, and phase B: acetonitrile. A gradient elution program ensured adequate retention, peak symmetry, and reproducible separation of the hydrophobic analyte within a single chromatographic run. Under the optimized conditions, 20-hydroxyecdysone exhibited stable retention and eluted at approximately 12.4 minutes.
DAD detection was performed at 245 nm, providing high sensitivity and selectivity for quantitative analysis. The method demonstrated excellent linearity, precision, accuracy, robustness, and repeatability across the validated concentration range. Importantly, no interference from excipients or formulation components was observed, confirming the method's selectivity in complex matrices.
The developed HPLC–DAD method is particularly relevant to nanomaterials research in the fields of medicine and pharmaceutical sciences. It is well-suited for the analysis of 20-hydroxyecdysone and other bioactive compounds incorporated into advanced lipid-based delivery systems. Due to its validated performance and matrix tolerance, the method serves as a reliable analytical tool for characterizing and quality controlling nanocarrier-based formulations that contain lipophilic active substances.
