Stability assessment of thymoquinone in cell culture systems: Effects of temperature, light exposure, and cell growth media

Thymoquinone (TQ) has significant in vitro therapeutic potential, but its chemical instability limits experimental reproducibility. This study investigates TQ thermal and photolytic degradation kinetics in various biological matrices to establish robust handling protocols. TQ solutions (10, 50, and 100 µM) were analyzed using a validated HPLC-UV method employing an Agilent 1260 Infinity HPLC system (Agilent Technologies, USA) with a Zorbax C18 column (4.6x150mm, 5 um). Photostability was assessed in a Suntest CPS+ chamber (Atlas, USA) per ICH Q1B guidelines (1.2 × 106 lux·h at 35°C for 9 h). Thermal stability was tracked for 168 h at 5°C, ambient, 25°C /60% RH, and 40°C /75% RH in serum-free and complete (10% fetal bovine serum, Elabscience, China) RPMI 1640 and DMEM. Photolytic stress caused extensive degradation, with 0.01% TQ remaining after 9h. Thermal degradation showed concentration-dependent, pseudo-first-order kinetics. Ambient exposure led to complete degradation within 24-96 h. Refrigerated storage (5°C) reduced degradation but recovery fell below 42% after 7 days. Matrix composition significantly influenced stability; DMEM and serum presence provided a partial protective effect, while RPMI formulations yielded less consistent recovery profiles. TQ exhibits high thermal and photolytic instability in cell media. ICH-compliant light triggers near-complete degradation, demanding immediate light protection during sample preparation. Although storage at 5°C improves short-term stability, substantial loss occurs over time. TQ stability is highly matrix-dependent, showing enhanced persistence in DMEM and serum-supplemented media. Strict light protection and minimized storage times are mandatory for reliable in vitro screening.