Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2'-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection

J Chromatogr B Biomed Sci Appl. 2000 Feb 11;738(2):311-7. doi: 10.1016/s0378-4347(99)00542-3.

Abstract

Exogenous and endogenous oxidants constantly cause oxidative damage to DNA. Since the reactive oxidants itself are not suitable for analysis, oxidized bases like 8-hydroxy-2'-deoxyguanosine (8OHdG) are used as biomarkers for oxidative stress, either in cellular DNA or as elimination product in urine. A simple, fast and robust analytical procedure is described for urinary 8OHdG as an indicator of oxidative damage in humans. The adduct was purified from human urine by applying a single solid-phase extraction step on LiChrolut EN. After evaporation of the eluate, the residue was resolved and an aliquote was injected into a HPLC system with a triple quadrupole mass spectrometer. The limit of detection was 0.2 ng ml(-1) (7 fmol absolute) when using one product ion as quantifier and two further product ions as qualifier. The coefficient of variation was 10.1% (n=5 at 2.8 ng ml(-1) urine). The sample throughput was about 50 samples a day. Thus, this method is more sensitive and much faster than the common method using HPLC with electrochemical detection. The results of a study with nine volunteers investigated at six time-points each over 5 days are presented. The mean excretion of 8OHdG was 2.1 ng mg(-1) creatinine (range 0.17-5.9 ng mg(-1) creatinine; 4 of 53 samples were below the LOD). A relatively large intra- (relative SD 66%) and inter-individual (relative SD 71%) variation in urinary 8OHdG excretion rates was found.

MeSH terms

  • 8-Hydroxy-2'-Deoxyguanosine
  • Adult
  • Biomarkers / urine*
  • Chromatography, High Pressure Liquid / methods*
  • Deoxyguanosine / analogs & derivatives*
  • Deoxyguanosine / urine
  • Female
  • Humans
  • Male
  • Mass Spectrometry
  • Middle Aged
  • Oxidative Stress*
  • Reproducibility of Results
  • Sensitivity and Specificity

Substances

  • Biomarkers
  • 8-Hydroxy-2'-Deoxyguanosine
  • Deoxyguanosine