Box–Behnken Design-Based Optimized UHPLC Method for the Analysis of Lipid-Based SNEDDS Ferrying Hydrocortisone and Thymoquinone

This study aimed to develop and validate a robust ultrahigh-performance liquid chromatography (UHPLC) method for the simultaneous quantification of hydrocortisone (HCT) and thymoquinone (TMQ) from self-nanoemulsifying drug delivery systems (SNEDDS). A Box-Behnken experimental design was applied to identify the impact of critical parameters, including the column temperature (20-40 °C), flow rate (0.2-0.4 mL/min), and buffer ratio (60-75%), on chromatographic responses. The optimized conditions comprised a column temperature of 20 °C, a flow rate of 0.2 mL/min, and a buffer ratio of 70.7%, achieving excellent peak resolution and asymmetry. The method demonstrated linearity (R² > 0.999) across the concentration range of 1-30 μg/mL for both analytes. Precision studies revealed relative standard deviations below 9.39% and 2.55% for HCT and TMQ, respectively, while accuracy ranged from 96.41% to 102.69% for HCT and from 97.83% to 106.21% for TMQ. The method exhibited high sensitivity with limits of detection of 0.203 and 0.129 μg/mL and limits of quantification of 0.615 and 0.392 μg/mL for HCT and TMQ, respectively. The method was robust, sensitive, and reproducible. The validated method was successfully applied to analyze HCT and TMQ contents with high recovery rates of 96.8 ± 2.53% and 101.5 ± 2.37%, respectively. The retention time for HCT was relatively shorter compared to that for TMQ, which may be attributed to various factors. This difference is more likely related to physicochemical properties such as hydrophobicity, polarity, and H-bonding capacity, together with chromatographic conditions, as both drugs are mainly un-ionized under the acidic mobile phase conditions. The optimized UHPLC method provides a reliable analytical tool for quality control and formulation development of SNEDDS.