The Box-Behnken Design for Optimizing HPLC Separation and Validation of Astilbin in Lysiphyllum strychnifolium Stems

The goal of the research was to use BBD, a productive RSM approach, to enhance the HPLC separation and validation of astilbin in Lysiphyllum strychnifolium stems. The percentage of acetonitrile (ACN), flow rate, and temperature were among the independent parameters that determined how much the chromatographic condition chosen from factor-level screens lowered the t of astilbin. The six dependent variables were t , PA, k', Rs, N, and As. The following HPLC settings were optimal for astilbin separation: 19% ACN at t ₀-t ₁₅, 0.8 mL/min flow rate, and 25°C temperature, resulting in a 26-min reduction in working time. This resulted in a separation success rate of 68.57%. Findings revealed the following sequence for t , PA, k', Rs, N, and As: 12.108 ± 0.010 min, 78,845,108 ± 420,267, 2.510 ± 0.003, 2.141 ± 0.024, 10,945 ± 80, and 0.991 ± 0.005. The limit of detection was 0.10 μg/mL, while the limit of quantitation was 0.20 μg/mL. The calibration curve was constructed using concentrations ranging from 0.39 to 50 μg/mL, with an R ² value of 0.9991, indicating excellent linearity. The intraday and interday precision RSD values were 0.069%-1.892% and 0.993%-3.229%, respectively. Recovery values were between 95.56% and 105.57%, confirming the method's accuracy. Astilbin was found at 175.51 ± 7.80 μg in L. strychnifolium stem extracts; its actual concentration was 3.51 ± 0.16%. The usefulness of astilbin as a chemical marker in L. strychnifolium stems may therefore be determined based on the criteria that have been established using this information.