Determination of 8-Oxoguanine in DNA by Gas Chromatography-Mass Spectrometry and HPLC-Electrochemical Detection: Overestimation of the Background Level of the Oxidized Base by the Gas Chromatography-Mass Spectrometry Assay

Abstract

Two analytical methods, one involving the combined use of reverse-phase HPLC and electrochemical detection (HPLC-EC) and one involving a mass spectrometric detection after gas chromatography separation (GC/MS), were developed for the detection of 8-oxoguanine in DNA. In order to obtain quantitative results, 2,6-diamino-8-oxopurine, whose chemical structure and electrochemical response are very similar to 8-oxoguanine, has been employed as an internal standard in the HPLC-EC assay. In the case of the GC/MS method, an isotopically stable (M + 4) 8-oxoguanine has been employed as an internal standard. Both methods are able to detect approximately 1 modification per 10(6) DNA bases. The background level of 8-oxoguanine in DNA as determined by GC/MS is approximately 50-fold higher than that determined by the HPLC-EC assay. The discrepancy between the two methods is due to an artifactual oxidation of guanine during the derivatization reaction as demonstrated by using pure guanine. The amount of 8-oxoguanine in guanine, determined by GC/MS, increases linearly with the time of derivatization, indicating that an oxidation occurs during the silylation reaction. Derivatization under nitrogen atmosphere reduces but does not suppress the artifactual oxidation. The amount of 8-oxoguanine in DNA, quantified by GC/MS, is comparable to that obtained by HPLC-EC when 8-oxoguanine is prepurified by HPLC or by immunoaffinity chromatography, prior to the silylation reaction. The artifactual formation of 8-oxoguanine during the derivatization reaction may explain, at least in part, why the values reported for 8-oxoguanine determination by GC/MS are generally about 1 order of magnitude higher than that determined by HPLC-EC. Prepurification of 8-oxoguanine from guanine is recommended in order to obtain reliable results by GC/MS which may be compared to HPLC-EC.

Effect of aging on mitochondrial and nuclear DNA oxidative damage in the heart and brain throughout the life-span of the rat

“…However, the 8-oxodG values reported in that study, which used a special micro-HPLC/mass spectrometry technique, were very high, between 100 and 1,500/10~dG (0.1-1.5% 8-oxodG/dG; 16). It is well known that mass spectrometry techniques have resulted in the past in artifactual 8-oxodG values (23,24). In contrast to those findings, no age-related increase was observed in heart 8-oxodG in mtDNA in mice from 12 to 26 months of age in a study reporting values of 6/ 10SdG in young adults (17).…”

Aging and the Accumulation of Oxidative Damage to DNA

“…2,2003 oxidation. For example, 8-oxodG levels obtained by GC/MS are considerably higher than those obtained using HPLC/EC [35]. Artifacts may occur during the derivatization of DNA acid-hydrolysates at high temperature prior to GC/MS analysis [36].…”

“…Several modified bases, such as 5-hydroxycytosine, 8-hydroxyadenine, 5-hydroxymethyluracil, and 5-formyluracil, as well as 8-oxoG, have been reported to be formed during the derivatization procedures for GC/MS [38]. It has been suggested that in the previous works, the derivatization under such improper conditions as the insufficient removal of oxygen might have been made [35,36,39]. Because LC/MS/MS and LC/MS do not require sample derivatization for assay, the oxidation of DNA is suppressed [34].…”

Development of microplate-based photoelectrochemical DNA biosensor array for high throughput detection of DNA damage

“…There are also a few established analytical methods for the identification and quantification of DNA damage products which can be used as molecular markers for genetic toxicity [6]. 8-Oxo-guanine (8-HO-dG) is a common product of oxidative DNA damage, and can be identified using HPLC separation followed by online electrochemical detection [7]. This method is very specific, but it requires relatively large and expensive equipments.…”