Quantitative measurement of radiation-induced base products in DNA using gas chromatography-mass spectrometry

Gas chromatography-mass spectrometry with selected-ion monitoring was used to study radiation-induced damage to DNA. Quantitative analysis of modified purine and pyrimidine bases resulting from exposure to ionizing radiation using this technique is dependent upon the selection of appropriate internal standards and calibration of the mass spectrometer for its response to known quantities of the internal standards and the products of interest. The compounds 6-azathymine and 8-azaadenine were found to be suitable internal standards for quantitative measurement of base damage in DNA. For the purpose of calibration of the mass spectrometer. relative molar response factors for intense characteristic ions were determined for the trimethylsilyl derivatives of 5-hydroxyuracil, thymine glycol, and 5,6-dihydrothymine using 6-azathymine, and for the trimethylsilyl derivatives of 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine using 8-azaadenine. Accurate measurement of the yield of radiation-induced modifications to the DNA bases is also dependent upon two chemical steps in which the purines and pyrimidines are released from the sugar-phosphate backbone and then derivatized to make them volatile for gas chromatography. The completeness of these reactions, in addition to assessing the stability of the modified DNA bases in acid and their trimethylsilylated derivatives over the time necessary to complete the experimental analysis was also examined. Application of this methodology to the measurement of radiation-induced base modification in heat-denatured, nitrous oxidesaturated aqueous solutions of DNA is presented.