Analytical Quality by Design (AQbD) Principles in Development and Validation of Stability‐Indicating RP‐HPLC Method for Simultaneous Estimation of Diltiazem HCl and Eugenol in Nanoethosomes

ABSTRACT The study developed and validated a robust reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method for a simultaneous estimation of Diltiazem HCl and Eugenol in nanoethosomes using analytical quality by design (AQbD) principles. The optimized method utilized a mobile phase of methanol and 0.1% Orthophosphoric acid (OPA) in a 50:50 % v/v ratio, with a flow rate of 1.0 mL/min and isocratic elution on a Phenomenex Luna C‐18 column (5 µm, 150 mm × 4.6 mm) at 30°C. This setup resulted in retention times of 6.838 min for Diltiazem HCl and 23.135 min for Eugenol. Validation followed International Council for Harmonization (ICH) Q2 (R1) guidelines, demonstrating excellent linearity with correlation coefficients of 0.9982 for Diltiazem HCl and 0.9991 for Eugenol across the 5–25 µg/mL range. The limits of detection (LOD) were 1.341 µg/mL for Diltiazem HCl and 0.960 µg/mL for Eugenol, with limits of quantification (LOQ) at 4.065 and 2.912 µg/mL, respectively. Stability testing under acidic, alkaline, oxidative, thermal, and photolytic conditions adhered to ICH Q1A (R2) and Q1B guidelines. This AQbD‐based method is effective for routine analysis of Diltiazem HCl and Eugenol in bulk and pharmaceutical dosage forms, fully complying with ICH standards.