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.
D001973:Bromodeoxyuridine / Q000032:analysis; D001976:Bromouracil / Q000032:analysis*; D004247:DNA / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry; D006868:Hydrolysis; D007202:Indicators and Reagents; D013941:Thymine / Q000032:analysis*
D007202:Indicators and Reagents; D001976:Bromouracil; D004247:DNA; D001973:Bromodeoxyuridine; D013941:Thymine