Quantitative determination of 8-hydroxy-2′-deoxyguanosine as a biomarker of oxidative stress in thalassemic patients using HPLC with an electrochemical detector

Abstract

A simple and robust HPLC method with electrochemical detection was developed for the quantitative determination of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a DNA damage product excreted in urine. Sample cleanup was carried out using solid-phase extraction (SPE) prior to chromatographic separation. 8-OHdG was well separated on an Eclipse XDB®-C18 column (150 × 4.6 mm i.d., 5 μm) with an Eclipse XDB®-C18 guard column (12 × 4.6 mm i.d., 5 μm). Two mobile phases containing methanol and 10 mM sodium formate (pH 4.5) at a ratio of 10: 90 and 50: 50 v/v, respectively, were used. The retention time of 8-OHdG was 9.8 ± 0.5 min. The recovery of 8-OHdG was found to be 97.2 ± 3.3% (n = 6). Intraday and interday precisions of the method were 4.0 ± 2.9% (n = 6) and 6.6 ± 1.7% (n = 6), respectively. The detection limit was 5 ng/mL. Preliminary investigation showed that the mean value of 8-OHdG, normalized with the amount of creatinine in the sample, from the thalassemic group was significantly higher than that from healthy subjects (211 ± 214 ng/mg creatinine vs. 31.4 ± 32.2 ng/mg creatinine, respectively), indicating oxidative stress.