A Gas Chromatography/Electron Capture/Negative Chemical Ionization High-Resolution Mass Spectrometry Method for Analysis of Endogenous and Exogenous N7-(2-Hydroxyethyl)guanine in Rodents and Its Potential for Human Biological Monitoring

A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry (GC/EC/NCI-HRMS) method was developed for quantitating N7-(2-hydroxyethyl)guanine (N7-HEG) with excellent sensitivity and specificity. [4,5,6,8-^13C4] -N7-HEG was synthesized, characterized, and quantitated using HPLC/electrospray ionization mass spectrometry (HPLC/ESI-MS) so it could serve as an internal standard. After being converted to its corresponding xanthine and derivatized with pentafluorobenzyl (PFB) bromide twice, the PFB derivative of N7-HEG was characterized using GC/EC/NCI-HRMS carried out at full scan mode. The most abundant fragment was at m/z 555, with a molecular formula of C21H9N4O3F10, resulting from the loss of one PFB group. By monitoring m/z 555.0515 (analyte) and m/z 559.0649 (internal standard), this assay demonstrated a linear relationship over a range of 1 fmol to 1 pmol of N7-HEG versus 20 fmol of [(13)C4] -N7-HEG on column. The limit of detection (LOD) for the complete assay was 600 amol (S/N = 5) injected on column. The variation of this assay was within 15% from 1 to 20 fmol of N7-HEG versus 2 fmol of [(13)C4] -N7-HEG with four replications for each calibration standard. Two hundred to three hundred micrograms of spleen DNA of control rats and mice and 100 (mu)g of spleen DNA of rats and mice exposed to 3000 ppm ethylene for 6 h/day for 5 days were analyzed using GC/EC/NCI-HRMS. The amounts of N7HEG varied from 0.2 to 0.3 pmol/(mu)mol of guanine in tissues of control rats. Ethylene-exposed animals had 5-15-fold higher N7-HEG levels than controls. This assay was able to quantitate N7-HEG in 25-30 (mu)g of DNA from human lymphocytes with excellent specificity. This was due in part to human tissues having 10-15-fold higher amounts of endogenous N7-HEG than rodents. These results show that this GC/EC/NCI-HRMS method is highly sensitive and specific and can be used in biological monitoring and molecular dosimetry and molecular epidemiology studies.