A high-performance liquid chromatographic method for the determination of 8-oxo-7,8-dihydro-2 '-deoxyguanosine in urine from man and rat

The renally excreted amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo(8)d G) is a potential marker of oxidative DNA damage by reactive oxygen species . We have developed a high-performance liquid chromatographic (HPLC) method to determine oxo(8)dG; in urine from humans and Wistar rats. First, 300 mu l of filtered urine is prefractionated by solid phase extraction (BAKER-BON D SPE C-18 Polar Plus column). Then, the HPLC separation of the fraction co ntaining oxo(8)dG is performed using four HPLC columns (two cation exchange and two Cls columns) in series with an automated column switching techniqu e. Quantification of oxo(8)dG is performed by electrochemical detection (Co ulochem II, ESA Inc.). Limit of detection was 0.4 nM oxo(8)dG. Recovery of oxo(8)dG added respectively in 11 or 8 concentration steps (range, 4-74 or 2-23 nM) to a pooled human or rat urine was 104.1 +/- 4.3 or 104.5 +/- 7.7% . Precision of sixfold analysis of a pooled human or rat urine carried out respectively on the same day was 2.2 or 2.4% relative standard deviation. N ormal excretion rates of oxo(8)dG in healthy adult humans (five females, si x males; body weight, 70.7 +/- 11 kg) and male Wistar rats (body weight, 30 9 +/- 13 g) were 281.7 +/- 179.1 and 333.2 +/- 47.4 pmol oxo(8)dG/ day/kg w eight, respectively. (C) 2000 Academic Press.