EFFECTS OF FORMIC-ACID HYDROLYSIS ON THE QUANTITATIVE-ANALYSIS OF RADIATION-INDUCED DNA-BASE DAMAGE PRODUCTS ASSAYED BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

Gas chromatography/mass spectrometry (GC/MS-SIM) is an excellent techn ique for performing both qualitative and quantitative analysis of DNA base damage products that are formed by exposure to ionizing radiation or by the interaction of intracellular DNA with activated oxygen spec ies. This technique commonly uses a hot formic acid hydrolysis step to degrade the DNA to individual free bases. However, due to the harsh n ature of this degradation procedure, the quantitation of DNA base dama ge products may be adversely affected. Consequently, we examined the e ffects of various formic acid hydrolysis procedures on the quantitatio n of a number of DNA base damage products and identified several facto rs that can influence this quantitation. These factors included (1) th e inherent acid stabilities of both the lesions and the internal stand ards; (2) the hydrolysis temperature; (3) the source and grade of the formic acid; and (4) the sample mass during hydrolysis. Our data also suggested that the N,O-bis (trimethylsilyl)trifluoroacetamide (BSTFA) derivatization efficiency can be adversely affected, presumably by tra ce contaminants either in the formic acid or from the acid-activated s urface of the glass derivatization vials. Where adverse effects were n oted, modifications were explored in an attempt to improve the quantit ation of these DNA lesions. Although experimental steps could be taken to minimize the influence of these factors on the quantitation of Som e base damage products, no single procedure solved the quantitation pr oblem for all base lesions. However, a significant improvement in the quantitation was achieved if the relative molecular response factor (R MRF) values for these lesions were generated with authentic DNA base d amage products that had been treated exactly like the experimental sam ples.