Optimization and validation of ultrasensitive GC-MS/MS method to measure oxidatively induced DNA damage products and role of antioxidants in oxidation mechanism.

Oxidation of DNA due to exposure to reactive oxygen species (ROS) is a major source of DNA damage. ROS induced damage to DNA plays an important role in some diseases such as various cancers, aging and neurodegenerative diseases. The detection of DNA oxidation products plays a major role in assessing the mutagenicity potential of specific exposure. The GC-MS/MS method was developed for the ultrasensitive determination of individual DNA damage products. The validation results revealed that the proposed method was reliable and sensitive. Multiple response surface methodology (MRSM) was used to optimize derivatization conditions of oxidatively DNA base damage products before GC-MS/MS analysis. The optimum derivatization conditions were determined as 40 min for derivatization time, 120 °C for derivatization temperature and 1.4 for BSTFA/pyridine ratio under nitrogen atmosphere. The effects of thymol, carvacrol and thymoquinone as antioxidants were investigated on oxidative DNA damage. The determination of the oxidatively induced DNA damage products was performed after adding DNA and antioxidants with different concentrations under oxidative stress. Eighteen DNA base damage products were analyzed simultaneously using GC-MS/MS. This study showed a significant decrease in the amount of DNA base damage products when the antioxidants were present in the medium.