Comprehensive analysis of the formation and stability of peroxynitrite-derived 8-nitroguanine by LC-MS/MS: Strategy for the quantitative analysis of cellular 8-nitroguanine.

Peroxynitrite is a major oxidizing and nitrating biological agent formed at sites of inflammation. Peroxynitrite can cause DNA damage and is thought to contribute to inflammation-related carcinogenesis. This study describes a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct determination of peroxynitrite-derived 8-nitroguanine (8-nitroGua) in DNA hydrolysates. This method exhibited a sensitive detection limit of 3 fmol and inter- and intraday imprecision of <10% and was applied to systemically examine the formation and stability of peroxynitrite-derived 8-nitroGua in different DNA substrates under various conditions. The 8-nitroGua formation was maximal at pH 8. The formation rate of 8-nitroGua in different DNA substrates decreased in the order of monodeoxynucleoside>single-stranded DNA>double-stranded DNA. A stability test revealed that the half-life for the depurination of 8-nitroGua from DNA was short and affected by both the temperature and DNA structure. When present in monodeoxynucleoside, the half-life of 8-nitroGua was estimated to be ~6min at 25°C and 2.3h at ~0°C. In single-stranded DNA, the half-life varied from 1.6h at 37°C to 533h at -20°C, whereas the half-life increased from 2.4h at 37°C to 1115h at -20°C in double-stranded DNA. We demonstrated that the measurement of 8-nitroGua in isolated DNA is not practicable because 8-nitroGua is unstable and lost during DNA extraction from cell. Therefore, we suggest that directly detecting cellular 8-nitroGua following nuclear membrane lysis is an alternative measure of the nitrative damage of nucleic acids, accounting for both DNA and RNA lesions within cells.