Urinary analysis of 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine by isotope-dilution LC-MS/MS with automated solid-phase extraction: Study of 8-oxo-7,8-dihydroguanine stability.

A highly sensitive quantitative method based on LC-MS/MS was developed to simultaneously and directly measure 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in urine. It was found that 8-oxoGua could be artifactually generated from 8-oxodGuo during the ionization process by both in-source thermolysis and collisionally induced dissociation. Our method applied a two-stage wash procedure in the online solid-phase extraction system that not only eliminated ion suppression but also prevented artifactual interference with 8-oxoGua by 8-oxodGuo by eluting the analytes individually. With the use of isotope internal standards, the detection limits of 8-oxoGua and 8-oxodGuo were estimated to be 30 and 3.5 fmol, respectively. The 8-oxoGua stability under common storage conditions was first investigated. Dissolved 8-oxoGua in NaOH (pH 12) was quite fragile and stable for only <1 day at room temperature. When pH and temperature were reduced, the 8-oxoGua stability at -20 degrees C was significantly increased to approximately 87 days in water (pH approximately 7) and approximately 112 days when diluted in 5% methanol. This method was further applied to measure urinary samples of healthy subjects. A molar ratio of 8-oxoGua to 8-oxodGuo of approximately 4.6 was found, supporting the hypothesis that oxidatively damaged DNA is primarily repaired by the base excision repair pathway.