Quantitative Stability Evaluation of Reconstituted Azacitidine Under Clinical Storage Conditions

Objectives: The aim of this study was to evaluate the stability of azacitidine (AZA) under clinical storage conditions (room temperature vs. refrigeration) to identify practical protocols that minimize waste and improve cost-effectiveness. Methods: AZA solutions (1 mg/mL) were stored at 23 ± 2 °C or 4 °C. Stability was assessed using a validated high-performance liquid chromatography (HPLC) method. Chromatographic separation was achieved on a Hypersil ODS C₁₈ column (250 mm × 4.6 mm, 5 μm) using an isocratic mobile phase of 50 mM potassium phosphate buffer (pH 7.0)-acetonitrile (98:2, v/v) at a flow rate of 1.0 mL/min, with UV detection at 245 nm and a 20 μL injection volume. The method demonstrated specificity for AZA and its main degradation product (DP), with LOD and LOQ of 12.56 μg/mL and 62.8 μg/mL, respectively. Linearity (R² = 0.9928), precision (RSD% < 5 for mid/high levels), and accuracy (mean recovery 96%) were established. Results: Azacitidine degraded rapidly at room temperature, with >85% loss within 24 h. In contrast, refrigeration at 4 °C significantly delayed degradation, with only ~26% loss observed over the same 24 h period. Chromatographic analysis confirmed the formation of a primary degradation product (tentatively identified as the open-ring hydrolytic species N-(formylamidino)-N'-β-D-ribofuranosylurea based on its chromatographic behavior and literature data), consistent with the known hydrolytic pathway. The applied HPLC-UV method offered an optimal balance of specificity and practicality for monitoring this main degradation trend under clinical storage conditions, distinguishing it from more complex techniques used primarily for structural elucidation. Conclusions: The pronounced instability of reconstituted AZA underscores the critical importance of strict adherence to immediate-use protocols. Refrigeration provides only a limited stability window. Based on our kinetic data, maintaining the reconstituted solution within an acceptable degradation limit (e.g., ≤10% loss) at 4 °C would require administration within a very short timeframe, supporting current handling guidelines to ensure therapeutic efficacy and minimize economic waste.