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 degrees C. Derivatization with 6-methoxynaphthylglyoxal at 40 degrees C for 30 min gave the strongest fluorescence product. The fluorescence derivatives from reaction with 6-methoxynaphthylglyoxal were separated on a Capcell Pak C18 SG 120A column (4.6 mm i.d. x 150 mm, 5 microm) with acetonitrile-5 mM 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 lambda(ex) 400 nm and lambda(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 microg/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 = 0.85x + 5.34. The proposed method was applied to the monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine from male heavy smokers.
Katayama
Masatoki
M
Department of Functional Bioanalysis, Meiji Pharmaceutical University, 2-522-1, Noshio, Kiyose, Tokyo 204-8588, Japan. mkatayama@my-pharm.ac.jp
Matsuda
Yoshifumi
Y
Kobayashi
Kensuke
K
Kaneko
Satoru
S
Ishikawa
Hiromichi
H
D000080242:8-Hydroxy-2'-Deoxyguanosine; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000652:urine; D005453:Fluorescence; D006037:Glyoxal / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D007118:Immunoassay; D007202:Indicators and Reagents; D008297:Male; D010658:Phenylglyoxal / Q000737:chemistry; D012907:Smoking / Q000652:urine
C513825:6-methoxy-2-naphthylglyoxal; D007202:Indicators and Reagents; D006037:Glyoxal; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine; D010658:Phenylglyoxal