HPLC method development, validation, and impurity characterization of a potent antitumor nucleoside, T-dCyd (NSC 764276)

Abstract

An HPLC method for the assay of an anticancer nucleoside, 4′-thio-2′-deoxycytidine (T−dCyd, NSC 764276), has been developed and validated. The stress testing of T-dCyd was carried out in accordance with ICH guidelines Q1A (R2) under acidic, alkaline, oxidative, thermolytic, and photolytic conditions. The separation of T-dCyd from its impurities and degradation products was achieved in 40min on a Luna® Phenyl-Hexyl column (150mm×4.6mm i.d., 3μm) with a gradient elution using ammonium phosphate buffer (pH 3.85) and methanol as the mobile phase. The gradient starts from 2% and ends at 80% of methanol. Detection is by UV at 282nm. LC–QTOF/MS was used to obtain mass data for characterization of impurities and degradation products. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.25–0.75mg/mL, r≥0.9998), accuracy (recovery 98.1–102.0%), precision (RSD≤1.5%), and sensitivity (LOD 0.1μg/mL). The developed method was suitable for the quality control and stability monitoring of the T-dCyd drug substance.