Kao Chi-Yu, Giese Roger W
Department of Pharmaceutical Sciences in the Bouve College of Health Sciences, Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.
Chem Res Toxicol. 2005 Jan;18(1):70-5. doi: 10.1021/tx049854d.
An improved method is presented, based on gas chromatography-electron capture mass spectrometry (GC-EC-MS), for measuring N7-(2'-hydroxyethyl)guanine (N7-HEG) in DNA from an in vivo sample. The method was used to detect this adduct in amounts of human DNA ranging from 0.07 to 11.5 microg isolated from granulocytes. In this method, the DNA is spiked with a stable isotope internal standard (N7-HEG-d4) and heated in water to release the adduct in a nucleobase form. After the adduct is extracted into 1-butanol, it is purified by reverse phase HPLC and derivatized with HONO, pentafluorobenzyl bromide, and pivalic anhydride. Further purification by silica solid phase extraction and reverse phase HPLC is done prior to injection into a GC-EC-MS. Relatively clean GC-EC-MS chromatograms result, contributing to the high sensitivity that is observed. In the samples tested, from 1.6 to 240 N7-HEG adducts in 10(7) nucleotides were observed, a 150-fold range.
本文介绍了一种基于气相色谱 - 电子捕获质谱法(GC - EC - MS)的改进方法,用于测量体内样品DNA中的N7 - (2'-羟乙基)鸟嘌呤(N7 - HEG)。该方法用于检测从粒细胞中分离出的0.07至11.5微克人DNA中的这种加合物。在此方法中,向DNA中加入稳定同位素内标(N7 - HEG - d4)并在水中加热,以释放出核碱基形式的加合物。加合物用1 - 丁醇萃取后,通过反相高效液相色谱法纯化,并用亚硝酸、五氟苄基溴和新戊酸酐进行衍生化。在注入GC - EC - MS之前,先通过硅胶固相萃取和反相高效液相色谱法进一步纯化。得到相对干净的GC - EC - MS色谱图,这有助于实现所观察到的高灵敏度。在所测试的样品中,在10⁷个核苷酸中观察到1.6至240个N7 - HEG加合物,范围达150倍。