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Abstract
An efficient method based on high-performance liquid chromatography with evaporative light scattering detection was developed for the separation and determination of four brassinolide analogs [24-epibrassinolide, (22S, 23S)-24-epibrassinolide, 28-homobrassinolide, and (22S, 23S)-28-epihomobrassinolide] without prior derivatization. The optimized analysis was carried out on a C18 reversed-phase column (150 mm × 4.60 mm, 3 µm) at 30°C using isocratic elution of acetonitrile and water (38:62, v/v). The drift tube temperature of the detector was 60°C and the auxiliary gas (nitrogen) pressure was 360 kPa. The regression equations revealed linear relationships (R2 = 0.9984–0.9994) within the test ranges. The limits of detection and quantification were in the ranges of 0.12 to 0.17 µg and 0.24 to 0.33 µg, respectively. The fully validated method was applied to quantify the active ingredient content in technical material and formulations and provides an alternative approach for quality control.