AQbD-Guided Stability-Indicating RP-HPLC-DAD Method for Curcuminoids in Commercial Capsules with Targeted Dye-Adulterant Screening

Abstract

A stability-indicating reversed-phase HPLC method with diode-array detection was developed for simultaneous determination of curcumin, demethoxycurcumin, and bisdemethoxycurcumin in commercial capsules, with targeted screening for metanil yellow and Sudan I. Analytical Quality by Design was applied through definition of an analytical target profile, risk assessment, scouting, and response-surface optimisation of initial ethanol content, aqueous-phase pH, and flow rate. Separation was achieved on a C18 column (150 × 4.6 mm, 3.5 µm) using 10 mM ammonium acetate at pH 3.2 and ethanol under gradient elution at 1.0 mL/min and 40 °C. Quantification was performed at 425 nm with full DAD acquisition from 240 to 550 nm. Bisdemethoxycurcumin, demethoxycurcumin, and curcumin eluted at approximately 4.85, 5.62, and 6.39 min, respectively. Calibration coefficients exceeded 0.99999, repeatability RSD values were 0.27–0.38%, and recoveries were 99.08–100.48%. The method remained robust to small changes in flow, wavelength, and pH. Forced degradation confirmed the stability order bisdemethoxycurcumin > demethoxycurcumin > curcumin, with alkaline and oxidative stress producing the greatest loss. Targeted dye spikes were identified by retention time and DAD spectral response. The commercial capsule contained 94.2% total curcuminoids, and neither targeted dye was detected. The ethanol-rich method gave an AGREE-style score of 0.71.