A high-throughput and sensitive methodology for the quantification of urinary 8-hydroxy-2'-deoxyguanosine: measurement with gas chromatography-mass spectrometry after single solid-phase extraction.
8-hydroxy-2'-deoxyguanosine (8OHdG) is a widely used biomarker for the measurement of endogenous oxidative DNA damage. A sensitive method for the quantification of 8OHdG in urine by single solid-phase extraction and GC-MS (gas chromatography with MS detection) using selective ion monitoring is described in the present study. After solid-phase extraction, samples are freeze-dried, derivatized by trimethylsilylation and analysed by GC-MS. The urinary 8OHdG was quantified using heavy isotope dilution with [18O]8OHdG. The recovery of 8OHdG after the solid-phase extraction ranged from 70 to 80% for a wide range of urinary 8OHdG levels. Using 1 ml of urine, the limit of quantification was >2.5 nM (2.5 pmol/ml) and the calibration curve was linear in the range 2.5-200 nM. This method was applied to measure 8OHdG in urine samples from 12 healthy subjects. The intra- and inter-day variations were <9%. Urinary 8OHdG levels in spot urine samples from four healthy subjects were also measured for 1 week and, again, the variation was small. The presence of H2O2 in urine did not cause artifactual formation of 8OHdG. Since this assay is simple, rapid, sensitive and reproducible, it seems suitable to be used as a routine methodology for the measurement of urinary excretion of 8OHdG in large population studies.
Department of Biochemistry, Faculty of Medicine, National University of Singapore, MD 7, Medical Drive, Singapore 117597, Singapore.
Hai-Shu
HS
Lin
Andrew M
AM
Jenner
Choon Nam
CN
Ong
Shan Hong
SH
Huang
Matthew
M
Whiteman
Barry
B
Halliwell
D000080242:8-Hydroxy-2'-Deoxyguanosine; D015415:Biomarkers / Q000652:urine; D003404:Creatinine / Q000652:urine; D004249:DNA Damage / Q000235:genetics; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000652:urine*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006147:Guanine / Q000031:analogs & derivatives* / Q000652:urine; D006151:Guanosine / Q000031:analogs & derivatives* / Q000652:urine; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D018384:Oxidative Stress / Q000235:genetics; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors
D015415:Biomarkers; D006151:Guanosine; C046215:8-hydroxyguanosine; C024829:8-hydroxyguanine; D006147:Guanine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003404:Creatinine; D006861:Hydrogen Peroxide; D003849:Deoxyguanosine