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

Abstract

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 μ M) results in increasing thymine substitution by BU over a range of 1–28%. Other important applications of this technique are mentioned.