Yen T Y, Christova-Gueoguieva N I, Scheller N, Holt S, Swenberg J A, Charles M J
Department of Environmental Sciences, University of North Carolina at Chapel Hill 27599-7400, USA.
J Mass Spectrom. 1996 Nov;31(11):1271-6. doi: 10.1002/(SICI)1096-9888(199611)31:11<1271::AID-JMS420>3.0.CO;2-J.
The need for specificity and sensitivity in the analysis of DNA adducts has led the development of GC/MS methods. Such methods require chemical derivatization (i.e. silylation, electrophore labelling), which can also bring its own sets of problems, including the production of artifacts, interferences and sample to sample variability in derivatization. To obviate such problems, a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method was developed to quantify N2,3-ethenoguanine (epsilon Gua), a promutagenic DNA adduct of vinyl chloride exposure. The response of epsilon Gua to isotopically labelled internal standard [13C4]epsilon Gua was linear (r2 = 0.999) and reproducible from 0.027 to 0.538 pmol microliter-1. We obtained an accuracy of 86 +/- 14% by analyzing chloroethylene oxide (CEO)-treated calf thymus DNA enriched with authentic epsilon Gua. The analysis of CEO-treated calf thymus DNA samples not enriched with authentic epsilon Gua provided a precision of 15%. The detection limits with a signal-to-noise ratio (S/N) 2.5:1 were obtained in the determination of authentic epsilon Gua at 5 fmol per injection. The detection limit obtained in the routine analysis of the biological samples was 50 fmol epsilon Gua with S/N = 3:1. The applicability of the method was established by determining epsilon Gua in rats treated with CEO by portal vein injection and an unexposed human liver. It was observed that the concentration of epsilon Gua in the rat livers increased with increase in dose and was inversely related to the time after, CEO exposure. This trend suggests rapid repair of the adduct in rat livers. In the human liver DNA sample, epsilon Gua was quantitated at 0.06 +/- 0.01 pmol mg-1 DNA.
对DNA加合物分析中特异性和灵敏度的需求推动了气相色谱/质谱（GC/MS）方法的发展。此类方法需要化学衍生化（即硅烷化、电泳标记），这也会带来其自身的一系列问题，包括假象的产生、干扰以及衍生化过程中样品间的差异。为避免这些问题，开发了一种液相色谱/电喷雾电离质谱（LC/ESI-MS）方法来定量N2,3-乙烯基鸟嘌呤（εGua），这是氯乙烯暴露产生的一种促突变DNA加合物。εGua对同位素标记的内标[13C4]εGua的响应呈线性（r2 = 0.999），在0.027至0.538 pmol微升-1范围内具有可重复性。通过分析用环氧氯乙烷（CEO）处理过的富含真实εGua的小牛胸腺DNA，我们获得了86±14%的准确度。对未富含真实εGua的CEO处理的小牛胸腺DNA样品的分析提供了15%的精密度。在每次进样5 fmol真实εGua的测定中，获得了信噪比（S/N）为2.5:1的检测限。在生物样品的常规分析中获得的检测限为50 fmol εGua，S/N = 3:1。通过测定经门静脉注射CEO处理的大鼠和未暴露的人肝脏中的εGua，确定了该方法的适用性。观察到大鼠肝脏中εGua的浓度随剂量增加而增加，并且与CEO暴露后的时间呈负相关。这种趋势表明大鼠肝脏中加合物的快速修复。在人肝脏DNA样品中，εGua的定量结果为0.06±0.01 pmol毫克-1 DNA。
