AQbD-Guided Stability-Indicating UHPLC Method for Simultaneous Determination of Major Withanolides in Ashwagandha Root Extract and Commercial Capsules

Abstract

A rapid, stability-indicating, and environmentally considerate ultra-high-performance liquid chromatographic (UHPLC) method was developed for simultaneous determination of withanoside IV, withanoside V, withaferin A, 12-deoxywithastramonolide, withanolide A, and withanolide B in Ashwagandha root extract and commercial capsules. Analytical Quality by Design (AQbD) was applied through definition of the analytical target profile, risk assessment, preliminary scouting, and response-surface optimisation of initial organic-phase strength, aqueous-phase pH, and flow rate. Separation was achieved on a C18 column (100 × 2.1 mm, 1.7 µm) using 5 mM ammonium formate at pH 3.2 and ethanol–acetonitrile (80:20, v/v) under gradient elution. The flow rate was 0.35 mL/min, detection was performed at 227 nm, and the total cycle time was 10.5 min. All six markers eluted between 3.62 and 6.71 min with minimum adjacent resolution above 2.0. Validation according to ICH Q2 (R2) demonstrated coefficients of determination above 0.9999, mean recoveries of 98.32–99.67%, and repeatability and intermediate-precision values below 0.5% RSD. Forced degradation under acidic, alkaline, oxidative, neutral, thermal, and photolytic conditions produced resolved degradant peaks with acceptable mass balance and peak purity. The reference root extract contained 2.60% total measured markers, whereas three commercial capsules contained 0.47–1.63%, demonstrating marked product-to-product variation. An Analytical Eco-Scale score of 84 and AGREE score of 0.79 supported the comparatively favourable environmental profile. The method is suitable for botanical standardisation, batch comparison, stability assessment, and routine quality control.