DESIGN OF EXPERIMENT (DOE) APPROACH FOR A SIMPLER HPLC TECHNIQUE ESTIMATING THYMOQUINONE FROM NIGELLA SATIVA SEEDS, COMMERCIAL FORMULATION, POLYMERIC NANOPARTICLES, AND ITS STABILITY INDICATION

To the best of our knowledge, the factorial design methodology has never been used to estimate Thymoquinone (TQ) using the reverse phase High-Performance Liquid Chromatography (RP-HPLC) method, making our work unique. The study aimed to create, verify and validate a simple stability-indicating analytical technique for quantitative detection of TQ in Ayurvedic formulations, black seed and polymeric nanoformulation using Design-experiment® (DOE) version 12. The technique was developed utilizing a Luna C18 HPLC column with a mobile phase of water: methanol (25:75, v/v), administered isocratic ally at 1 ml/min, at 253 nm. The validation parameters were carried out in compliance with ICH criteria using the Quality by Design (QbD) approach. The devised technique was shown to be specific, linear (R2 > 0.999) across the specified concentration range of 0.5 to 16 µg/ml, with detection and quantification limits of 0.02 and 0.08 µg/ml, respectively. The applicability of the new approach was evaluated using the TQ entrapped polymeric nanoparticle. TQ entrapment efficiency for prepared nanoformulation was found to be 82.87 percent. The experimental model was significant (P 0.0001), as evidenced by purposeful adjustments in the approach examined using analysis of variance. The approach was used to calculate the amount of TQ in black seed, and commercially available formulations were discovered to be equivalent to the labeled concentration. A deeper understanding of the parameters that drive chromatographic separation is achieved using the QbD approach. The proposed approach proved fast, cheap and exact for quantifying TQ in bulk and nanoparticulate systems.