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

• Published in: Free radical biology & medicine 2010, Vol.48 (1), p.89-97
• Main Authors: Hu, Chiung-Wen, Chao, Mu-Rong, Sie, Ciao-Han
• Format: Article
• Language: English
• Subjects: 8-OxoGua; Adult; Chromatography, High Pressure Liquid; Deoxyguanosine - analogs & derivatives; Deoxyguanosine - chemistry; Deoxyguanosine - urine; Female; Free radicals; Guanine - analogs & derivatives; Guanine - chemistry; Guanine - urine; Humans; LC–MS/MS; Male; Nitrogen Isotopes; Online SPE; Oxidatively damaged DNA; Reference Values; Sensitivity and Specificity; Solid Phase Extraction; Stability; Tandem Mass Spectrometry; Time Factors
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2009.10.029

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°C was significantly increased to ∼ 87 days in water (pH ∼ 7) and ∼ 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 ∼ 4.6 was found, supporting the hypothesis that oxidatively damaged DNA is primarily repaired by the base excision repair pathway.