Prevention of artifactual oxidation in determination of cellular 8-oxo-7,8-dihydro-2′-deoxyguanosine by isotope-dilution LC–MS/MS with automated solid-phase extraction

Abstract

A highly sensitive quantitative method based on LC–MS/MS was developed to directly measure 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 2′-deoxyguanosine (dG) in crude DNA hydrolysates. With the use of isotopic internal standards and online solid-phase extraction (SPE), this method has overcome the artifactual response often observed during electrospray ionization by optimizing the washing conditions of online SPE to remove excess dG and allows 8-oxodG and dG to be accurately and simultaneously monitored by mass spectrometry. The detection limit of this method was estimated as 1.8 fmol for 8-oxodG. With this method, we further investigated the artifactual oxidation that occurred during concentration and purification of the DNA hydrolysates, commonly used before sample analysis. Our results demonstrated that drying under vacuum or purification with C18 cartridges led to a significant increase in the measured 8-oxodG by 6.8–30 8-oxodG/10 6 dG. The artifactual formation of 8-oxodG can be reduced only by adding desferrioxamine (DFO) and not 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). However, DFO still failed to offer complete protection against oxidation during DNA hydrolysate concentration and purification. Therefore, to effectively prevent the artifacts formed during workup, the simplest approach is to use a direct measurement method involving an online enrichment/purification technique as proposed in this study.