Simultaneous determination of thymine and 5-bromouracil in DNA hydrolysates using gas chromatography-mass spectrometry with selected-ion monitoring.

The gas chromatographic-mass spectrometric method using selected-ion monitoring (GC-MS-SIM) described here quantitatively determines the amount of DNA thymine replacement by 5-bromouracil (BU) after exposure to 5-bromo-2'-deoxyuridine (BUDR) in as few as 10(5) cells. DNA is extracted, enzymatically hydrolyzed, the nucleic acid bases (with added internal standards, 5-iodouracil and 5-chlorouracil) are extracted into ethyl acetate, concentrated and derivatized with bis(trimethylsilyl)trifluoroacetamide. Thymine and BU are then quantitated by GC-MS-SIM. Response is linear to thymine over the range of 100-2000 ng per sample and BU of 1.3-52 ng per sample with a coefficient of variation of less than 10% and an accuracy for seeded samples within 8% of theoretical value. With V79 cells in culture, exposure to increasing BUDR concentrations (0.03-1.0 microM) results in increasing thymine substitution by BU over a range of 1-28%. Other important applications of this technique are mentioned.