Stetson P L, Maybaum J, Shukla U A, Ensminger W D
J Chromatogr. 1986 Feb 14;375(1):1-9. doi: 10.1016/s0378-4347(00)83686-5.
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.
本文所述的采用选择离子监测的气相色谱 - 质谱法（GC-MS-SIM）可在仅10⁵个细胞中定量测定暴露于5-溴-2'-脱氧尿苷（BUDR）后DNA中胸腺嘧啶被5-溴尿嘧啶（BU）取代的量。提取DNA，进行酶解，将核酸碱基（添加内标5-碘尿嘧啶和5-氯尿嘧啶）萃取到乙酸乙酯中，浓缩并用双（三甲基硅基）三氟乙酰胺进行衍生化。然后通过GC-MS-SIM对胸腺嘧啶和BU进行定量。对于每个样品中胸腺嘧啶，在100 - 2000 ng范围内响应呈线性，对于每个样品中BU，在1.3 - 52 ng范围内响应呈线性，变异系数小于10%，对于接种样品的准确度在理论值的8%以内。对于培养中的V79细胞，暴露于不断增加的BUDR浓度（0.03 - 1.0 microM）会导致胸腺嘧啶被BU取代的比例在1 - 28%范围内增加。还提到了该技术的其他重要应用。
