Monitoring of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine in urine by high‐performance liquid chromatography after pre‐column derivatization with glyoxal reagents

A new, simple and sensitive pre‐column fluorescence derivatization high‐performance liquid chromatographic method for the determination of the oxidative DNA stress marker, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine, was developed. Solid‐phase extraction using an Oasis HLB cartridge avoided troublesome sample preparation steps, interference from charged species and frequent and essential electrode maintenance in electrochemical procedures. 8‐Oxo‐7,8‐dihydro‐2′‐deoxyguanosine and other guanine compounds were selectively derivatized with glyoxal reagents (phenylglyoxal, 3,4‐methylenedioxyglyoxal, 2‐naphtylglyoxal and 6‐methoxynaphthylglyoxal) at 40–60°C. Derivatization with 6‐methoxynaphthylglyoxal at 40°C for 30 min gave the strongest fluorescence product. The fluorescence derivatives from reaction with 6‐methoxynaphthylglyoxal were separated on a Capcell Pak C 18 SG 120A column (4.6 mm i.d. × 150 mm, 5 µm) with acetonitrile–5 m M phosphate buffer (pH 6.0; 3:7, v/v) as mobile phase. The detection wavelength of the fluorescence derivative of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine was λ ex 400 nm and λ em 510 nm. The detection limit of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine was 1 ng/mL using 50 mL of urine. The calibration graphs were linear up to 30 µg/mL for 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine. The relative standard deviation of 20 ng/mL of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine was 7.0%. The proposed method was compared with the enzymatic ELISA 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine analysis method (8‐OH‐dG Check, JaICA, Shizuoka, Japan). The correlation coefficient was 0.79 ( n = 20) and y = 0.85 x + 5.34. The proposed method was applied to the monitoring of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine in urine from male heavy smokers. Copyright © 2006 John Wiley & Sons, Ltd.