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 (oxo8dG) is a potential marker of oxidative DNA damage by reactive oxygen species. We have developed a high-performance liquid chromatographic (HPLC) method to determine oxo8dG in urine from humans and Wistar rats. First, 300 μl of filtered urine is prefractionated by solid phase extraction (BAKERBOND SPE C18 Polar Plus column). Then, the HPLC separation of the fraction containing oxo8dG is performed using four HPLC columns (two cation exchange and two C18 columns) in series with an automated column switching technique. Quantification of oxo8dG is performed by electrochemical detection (Coulochem II, ESA Inc.). Limit of detection was 0.4 nM oxo8dG. Recovery of oxo8dG 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 oxo8dG 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 oxo8dG/day/kg weight, respectively.