Organotellurium compounds present analytical challenges due to their chemical reactivity and potentially limited stability. This study aimed to develop and validate a stability-indicating high-performance liquid chromatography method with ultraviolet detection (HPLC-UV) for oxichloro telluro anisole (Te18OH), integrating Analytical Quality by Design (AQbD), forced degradation, measurement uncertainty, and computational chemistry. Chromatographic conditions were optimized using a 20-run central composite design to investigate the effects of methanol proportion, trifluoroacetic acid concentration, and column temperature. The optimized method employed a C18 column, methanol–water containing 0.20% trifluoroacetic acid (20:80, v/v), a flow rate of 0.6 mL/min, ultraviolet detection at 247 nm, and a column temperature of 35 °C. Regression models were statistically significant (p < 0.0001), and Monte Carlo simulations indicated a probability exceeding 99% of simultaneously meeting the predefined chromatographic acceptance criteria across the evaluated operating conditions. The method demonstrated satisfactory precision, with relative standard deviations of 0.94% for repeatability and 0.69% for intermediate precision, and a combined measurement uncertainty of 2.14%. Forced degradation studies showed that oxidative stress was the only condition producing substantial degradation, generating one major product corresponding to 13.90% relative recovery, with chromatographic resolution greater than 2.0. Density functional theory calculations and in silico degradation predictions supported a plausible oxidative transformation involving the tellurium–chlorine bond, although the degradation product structure could not be definitively confirmed. This integrated analytical and computational strategy provides a robust approach for characterizing Te18OH stability and offers a framework for investigating other organotellurium compounds.
