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

Abstract The renally excreted amount of 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo 8 dG) 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 μl of filtered urine is prefractionated by solid phase extraction (BAKERBOND SPE C 18 Polar Plus column). Then, the HPLC separation of the fraction containing oxo 8 dG is performed using four HPLC columns (two cation exchange and two C 18 columns) in series with an automated column switching technique. Quantification of oxo 8 dG is performed by electrochemical detection (Coulochem 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. Normal excretion rates of oxo 8 dG in healthy adult humans (five females, six males; body weight, 70.7 ± 11 kg) and male Wistar rats (body weight, 309 ± 13 g) were 281.7 ± 179.1 and 333.2 ± 47.4 pmol oxo 8 dG/day/kg weight, respectively.